################################################################################## # RLIMS-P Benchmarking Data Set for Retrieving Papers of Protein Phosphorylation # # Information (total 370) # # # # Positive set (Pos-): 110 # # Negative set (Neg-): 260 # # # # Each record is referenced to PMID and PIR protein ID. # # # # January 24, 2005 http://georgetown.edu/iprolink/ # ################################################################################## Pos-1. PMID:8125092 | PIR:A40936 TI - Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38. AB - Oncoprotein 18 (Op18) has been independently identified due to its increased phosphorylation in response to external signals and its up-regulated expression in acute leukemia. We have identified two serine residues of Op18 that are phosphorylated after triggering by the T cell antigen receptor. One of these residues, Ser25, was shown to be a likely substrate for the mitogen-activated protein (MAP) kinase, while the other residue, Ser16, was shown to be phosphorylated in response to increased intracellular calcium. Our previous site-mapping studies of Op18 also revealed that basal phosphorylation of Op18 is mainly located on Ser38, which was found to be the primary in vitro phosphorylation site of p13suc1-precipitated cdc2 kinase activities. These findings raised the possibility that Op18 may be a substrate for both receptor-regulated calcium-induced protein kinases and the MAP kinase family, as well as being a substrate for the cell-cycle-regulated cdc2 kinase family. In the present report we have performed site-mapping studies of cell-cycle-regulated fluctuations of Op18 phosphorylation. The results reveal that S-phase progression of a synchronised leukemic T cell line is associated with increased phosphorylation of both the Ser25 and Ser38 residues. Moreover, during mitosis, a burst of phosphorylation was observed and at this stage of the cell cycle a major fraction of Op18 was phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at an unidentified C-terminal residue. In vitro phosphorylation experiments, employing two distinct members of the cdc2 kinase family, were consistent with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or p33-cdk2, was found to be the same as the ratio observed in intact cells during all phases of the cell cycle. These findings suggest that Op18 may be a physiological substrate for several members of the cdc2 kinase family during both the S-phase and the mitotic phase of the cell cycle. SO - Eur J Biochem 1994 Mar 1;220(2):359-68. Pos-2. PMID:8404852 | PIR:I38344 TI - Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts. AB - Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis. Heidelberg. SO - EMBO J 1993 Oct;12(10):3827-34. Pos-3. PMID:2846551 | PIR:A31780 TI - Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites. AB - Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro . The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C. SO - J Biol Chem 1988 Nov 15;263(32):16796-801. Pos-4. PMID:2492519 | PIR:HHHU84 HHHU86 TI - Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II. AB - Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp 90s begin with proline; the initial methionine residue is removed. The alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4 to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form. The purified hsp 90 mixture contains 2 mol of phosphate/mol of polypeptide. Both hsp 90 proteins are phosphorylated at two homologous sites. For the alpha protein, these sites correspond to serine 231 and serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation sites is absent from the beta protein. The sequence between phosphorylation sites of both hsp 90s is predicted to have alpha helical structure. Dephosphorylated hsp 90 is phosphorylated at both sites by casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no other hsp 90 residues were phosphorylated by casein kinase II in vitro. SO - J Biol Chem 1989 Feb 15;264(5):2431-7. Pos-5. PMID:3277962 | PIR:R3RTS6 TI - The primary structure of rat ribosomal protein S6. AB - The amino acid sequence of rat ribosomal protein S6, the major phosphoprotein in the organelle, was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the sequence of amino acids in portions of the protein. Ribosomal protein S6 contains 249 amino acids and has a molecular weight of 28,683. The protein has 15 seryl residues; 7 are located in the carboxyl-terminal sequence of 15 amino acids and probably include most if not all of the residues that are phosphorylated. There are related repeated sequences of 10 amino acids each that occur at four separate positions in S6 and that are very basic. Rat S6 is homologous to Saccharomyces cerevisiae S10 (the extent of the identity is 75%) and most likely also to Schizosaccharomyces pombe S6. Illinois 60637. SO - J Biol Chem 1988 Feb 25;263(6):2891-6. Pos-6. PMID:2899026 | PIR:WHRTY TI - Role of the N-terminus of rat pheochromocytoma tyrosine hydroxylase in the regulation of the enzyme's activity. AB - Activation of rat pheochromocytoma tyrosine hydroxylase by limited tryptic proteolysis was investigated. The modifications produced upon the enzyme's structure were analyzed with the use of sodium dodecyl sulfate/polyacrylamide gel electrophoresis and tyrosine hydroxylase activity was measured all through the digestion. During the proteolysis the activity of tyrosine hydroxylase was elevated threefold at the same time as a 56-kDa tryptic fragment was formed. When the enzyme was phosphorylated, at its N-terminal region, by a kinase copurified with tyrosine hydroxylase, the major 56-kDa species did not appear to be phosphorylated on the autoradiograph, suggesting that it was derived from the native subunit by cleavage of the N-terminal of the protein. The reactivity of the 2/40/15 anti-(tyrosine hydroxylase) monoclonal antibody with the N-terminal of tyrosine hydroxylase was also investigated, using the Western-blot technique. This antibody reacted with the 62-kDa hydroxylase subunit but not with the 60-kDa tryptic fragment; the amino acid sequences of these two species showed that the 60-kDa fragment lacked the first 16 N-terminal amino acids of the native molecule. These results suggest that the N-terminal region of tyrosine hydroxylase is apparently responsible for an inhibition of the hydroxylase activity and that the first N-terminal amino acids of the hydroxylase are necessary for the recognition of the enzyme by its antibody. SO - Eur J Biochem 1988 Jul 1;174(4):685-90. Pos-7. PMID:8491187 | PIR:TPHUN1 TI - Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation. AB - The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations. SO - EMBO J 1993 May;12(5):1937-46. Pos-8. PMID:2377895 | PIR:KIRTC2 TI - Autophosphorylation of protein kinase C at three separated regions of its primary sequence. AB - The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation. Berkeley 94720. SO - Science 1990 Jul 27;249(4967):408-11. Pos-9. PMID:6698958 | PIR:R3RTS6 TI - Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of the major phosphorylation sites for cAMP-dependent protein kinases. AB - Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes. SO - J Biol Chem 1984 Feb 25;259(4):2084-91. Pos-10. PMID:278975 | PIR:TMRBA TI - Specific phosphorylation at serine-283 of alpha tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle. AB - Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32P]orthophosphate, was fractionated into two components, alpha and beta, on a CM-cellulose column. Radioactivity was associated only with the alpha component. A single phosphorylation site was located at serine-283 (pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal alpha and cardiac tropomyosin. The presence of covalently bound phosphate in alpha tropomyosin and its absence in the beta component of rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino acid sequences around the phosphorylation sites of frog and rabbit tropomyosin are identical. Because this sequence is not similar to any other known phosphorylation site in proteins, this indicates the existence of either specific kinase or phosphatase that can distinguish between alpha and beta tropomyosins. In a model proposed for the head-to-tail overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue could form a salt linkage with lysine-6 on one side of the overlap region and another with lysine-12 on the other side. This would predict a difference in the stability of polymers of phosphorylated and nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin. SO - Proc Natl Acad Sci U S A 1978 Aug;75(8):3588-92. Pos-11. PMID:2203790 | PIR:A38379 TI - Amino acid and cDNA sequence of bovine phosducin, a soluble phosphoprotein from photoreceptor cells. AB - Vertebrate photoreceptor cells contain a soluble phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the GTP-binding protein, transducin. Light-induced changes in cyclic nucleotide levels modulate the phosphorylation of phosducin by protein kinase A. The complete amino acid sequence of purified phosducin from bovine retinas was determined by Edman degradation from overlapping polypeptides derived from enzymatic digestion by trypsin and Staphylococcus aureus V8 protease or from chemical degradation by cyanogen bromide. Excluding the unidentified group which blocks the NH2 terminus, phosducin contains 245 amino acids with a calculated molecular weight of 28,185 and isoelectric point of pH 4.5. Phosducin is enriched with acidic and sulfur-containing amino acids, having 32 glutamic acid, 16 aspartic acid, 9 methionine, and 5 cysteine residues. It also contains 24 serine and 8 threonine residues, of which only serine 73 is located within a consensus phosphorylation sequence (-RKMS(P)QV-) for cyclic nucleotide-dependent protein kinase. Secondary structure analysis predicts the presence of 62% alpha-helix, 22% beta-sheet, and 16% random coil, with eight turns. Computer-aided searches of protein data banks revealed no apparent homology to any sequenced protein except that coded by a MEKA cDNA clone (Kuo, C-H., Akiyama, M., and Miki, N. (1989) Mol. Brain Res. 6, 1-10) which deviates from the confirmed phosducin sequence in the last 15 amino acids. Sequence analysis of a cDNA clone for bovine retinal phosducin confirmed that the MEKA clone deviation resulted from an unidentified cDNA guanosine nucleotide, a shifted reading frame and a premature stop codon. SO - J Biol Chem 1990 Sep 15;265(26):15867-73. Pos-12. PMID:2117608 | PIR:A33369 TI - Phosphate groups as substrate determinants for casein kinase I action. AB - Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates. Indianapolis 46223. SO - J Biol Chem 1990 Aug 25;265(24):14264-9. Pos-13. PMID:2226863 | PIR:TPHUIC TPRBIC TI - A common motif of two adjacent phosphoserines in bovine, rabbit and human cardiac troponin I. AB - From rabbit and human cardiac troponin I N-terminal mono and bisphosphorylated peptides were isolated which were obtained from Lys-C proteinase digests. Two adjacent phosphoserine residues could be localized in each phosphopeptide following further tryptic digestion. The previously published sequence of rabbit cardiac troponin I had to be corrected. Two adjacent phosphoserine residues are a common motif in the very similar sequences of bovine, rabbit and human cardiac troponin I. The N-terminal sequences are: AcADRSGGSTAG DTVPAPPPVR RRS(P)S(P)ANYRAY ATEPHAK (bovine), AcADESTDA-AG EARPAPAPVR RRS(P)S(P)ANYRAY ATEPHAK (rabbit), (Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RRS(P)S(P)-NYRAY ATEPHAK (human). Supramolekularer Systeme, Ruhr-Universitat Bochum, FRG. SO - FEBS Lett 1990 Oct 29;273(1-2):41-5. Pos-14. PMID:8647113 | PIR:S57631 TI - The guanine-nucleotide-exchange complex (EF-1 beta gamma delta) of elongation factor-1 contains two similar leucine-zipper proteins EF-1 delta, p34 encoded by EF-1 delta 1 and p36 encoded by EF-1 delta 2. AB - We have cloned and sequenced a Xenopus cDNA referred to as EF-1 delta 2. The cDNA is homologous to EF-1 delta 1 encoding for EF-1 delta a protein of the guanine-nucleotide exchange complex of elongation factor-1 (EF-1). The protein sequence deduced from the cDNA, contains the two characteristic features of EF-1 delta protein, the leucine-zipper domain and the guanine-nucleotide exchange domain. In vitro and in vivo translation leads to the production of a 36-kDa protein from EF-1 delta and a 34-kDa protein from EF-1 delta 1. The clone EF-1 delta 2 therefore encodes for authentic p36 protein of EF-1 beta gamma delta complex, while EF-1 delta 1 encodes for a newly characterised p34 protein of the leucine zipper family. Both EF-1 delta proteins are simultaneously present in oocytes extracts, at a molecular ratio around 1:10 for p34 versus p36 proteins. Both are associated in a macromolecular structure that is greater than 750 kDa upon gel filtration. The two proteins are targets for Cdc2 kinase in meiotic maturation. Curie, France. SO - Eur J Biochem 1996 May 1;237(3):685-90. Pos-15. PMID:1846781 | PIR:TVHUJN TI - Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. AB - In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation. California 92037. SO - Cell 1991 Feb 8;64(3):573-84. Pos-16. PMID:1695717 | PIR:DVHUCF TI - A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. AB - The gene responsible for cystic fibrosis (CF) has recently been identified and is predicted to encode a protein of 1,480 amino acids called the CF transmembrane conductance regulator (CFTR). Several functional regions are thought to exist in the CFTR protein, including two areas for ATP-binding, termed nucleotide-binding folds (NBFs), a regulatory (R) region that has many possible sites for phosphorylation by protein kinases A and C, and two hydrophobic regions that probably interact with cell membranes. The most common CF gene mutation leads to omission of phenylalanine residue 508 in the putative first NBF, indicating that this region is functionally important. To determine whether other mutations occur in the NBFs of CFTR, we determined the nucleotide sequences of exons 9, 10, 11 and 12 (encoding the first NBF) and exons 20, 21 and 22 (encoding most of the second NBF) from 20 Caucasian and 18 American-black CF patients. One cluster of four mutations was discovered in a 30-base-pair region of exon 11. Three of these mutations cause amino-acid substitutions at residues that are highly conserved among the CFTR protein, the multiple-drug-resistance proteins and ATP-binding membrane-associated transport proteins. The fourth mutation creates a premature termination signal. These mutations reveal a functionally important region in the CFTR protein and provide further evidence that CFTR is a member of the family of ATP-dependent transport proteins. Baltimore, Maryland 21205. SO - Nature 1990 Jul 26;346(6282):366-9. Pos-17. PMID:8388392 | PIR:A45100 TI - Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. AB - Mitogen-induced signal transduction is mediated by a cascade of protein phosphorylation and dephosphorylation. One of the immediate responses of mitogen stimulation is the activation of a family of protein kinases known as mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK). MEK (MAP kinase or ERK kinase) is the immediate upstream activator kinase of ERK. Two cDNAs, MEK1 and MEK2, were cloned and sequenced. MEK1 and MEK2 encode 393 and 400 amino acid residues, respectively. The human MEK1 shares 99% amino acid sequence identity with the murine MEK1 and 80% with human MEK2. Both MEK1 and MEK2 were expressed in Escherichia coli and shown to be able to activate recombinant human ERK1 in vitro. The purified MEK2 protein stimulated threonine and tyrosine phosphorylation on ERK1 and concomitantly activated ERK1 kinase activity more than 100-fold. The recombinant MEK2 showed lower activity as an ERK activator as compared with MEK purified from tissue. However, the recombinant MEK2 can be activated by serum-stimulated cell extract in vitro. MEKs, in a manner similar to ERKs, are likely to consist of a family of related proteins playing critical roles in signal transduction. 48109. purification/metabolism SO - J Biol Chem 1993 May 25;268(15):11435-9. Pos-18. PMID:3114005 | PIR:QFPGM S02571 TI - Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H. AB - Diagonal fingerprinting allows the specific purification of those tryptic peptides which change electrophoretic mobility due to a dephosphorylation step introduced after the first dimension. Nine tryptic peptides from the tail domain of porcine neurofilament M protein identify a minimum of 6 phosphorylated serines. Unexpectedly, four of the nine peptides characterize a region of degenerate repetitive sequences. Results on neurofilament H tail, although less complete, yield longer sequences of degenerate repetitive character. Here, all serines present appear to be contained in a lysine-serine-proline unit. This motif also occurs in some but not all M peptides. We suggest that degenerate repetitive sequences in neurofilament M and H tails have a high species-specific drift. SO - FEBS Lett 1987 Sep 14;221(2):403-7. Pos-19. PMID:3112144 | PIR:DCECIS TI - Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. AB - The control of isocitrate dehydrogenase through phosphorylation is necessary for growth of Escherichia coli on acetate as the sole carbon source. To understand the mechanism by which phosphorylation inactivates isocitrate dehydrogenase, the sequence of icd, the isocitrate dehydrogenase structural gene, was determined and this information was used to construct mutants at the site of phosphorylation. Introduction of a negatively charged aspartate for the serine that is phosphorylated completely inactivates isocitrate dehydrogenase. Substitution of the serine with other amino acids results in a partially active enzyme in which both maximal velocity and interaction with substrates has been altered. Neither threonine nor tyrosine, when substituted for the serine at the phosphorylation site, is detectably phosphorylated by isocitrate dehydrogenase kinase. SO - J Biol Chem 1987 Aug 5;262(22):10422-5. Pos-20. PMID:2040274 | PIR:BGSH TI - Nuclear transition protein 1 from ram elongating spermatids. Mass spectrometric characterization, primary structure and phosphorylation sites of two variants. AB - The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S-carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys-C. The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39. From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double-stranded DNA. The presence of three phosphorylatable serine residues in the very conserved sequence 29-42 is another argument for the involvement of this region in the interaction with DNA. Scientifique, Universite de Lille II, France. SO - Eur J Biochem 1991 May 23;198(1):13-20. Pos-21. PMID:2122883 | PIR:TIHUGK TI - Endogenous phosphorylation of the lipoprotein-associated coagulation inhibitor at serine-2. AB - Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor VIIa-tissue factor (TF), presumably by forming a quaternary Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2 cells grown in the presence of [32P]orthophosphate was observed to be covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase resulted in an almost complete removal of the radiolabel. Phosphoamino acid analysis of the purified 32P-LACI established that the phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI contains a cluster of acidic residues C-terminal to the serine-2 residue. Such a site is characteristic of the sites phosphorylated by casein kinase II (CKII) in protein substrates. Edman degradation of endogenously labelled 32P-LACI revealed that the serine-2 residue was a major site of phosphorylation. Phosphorylation of purified LACI by bovine CKII was observed to occur in vitro; amino acid sequence analysis demonstrated that CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI expressed from mouse C127 fibroblasts transfected using a bovine-papilloma-virus expression vector was found to be endogenously phosphorylated. By using site-directed mutagenesis, an altered form of LACI was produced in which the serine-2 residue had been changed to alanine. This altered LACI, although expressed in similar quantity to the wild-type LACI, was not detectably phosphorylated. Using the altered LACI in functional studies demonstrated that a serine residue at position 2, and thus the phosphorylation of this site, was not essential for LACI's inhibition of Xa and VIIa-TF activities. Jewish Hospital, St. Louis, MO 63110. SO - Biochem J 1990 Sep 15;270(3):621-5. Pos-22. PMID:6290217 | PIR:PZRB1 TI - Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle. AB - The complete primary structure of protein phosphatase inhibitor-1 has been determined. The protein consists of a single polypeptide chain of 165 residues, molecular weight 18640. The threonine residue that must be phosphorylated for activation is at position 35 and the active cyanogen bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but this phosphoserine residue does not appear to influence the activity of inhibitor-1. SO - Eur J Biochem 1982 Aug;126(2):235-46. Pos-23. PMID:3185734 | PIR:QRECCS TI - Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. AB - A cascade of protein phosphorylation, initiated by autophosphorylation of the CheA protein, may be important in the signal transduction pathway of bacterial chemotaxis. A proteolytic fragment of CheA cannot autophosphorylate, but can still transfer phosphate to proteins that generate excitation and adaptation signals. The site of CheA phosphorylation is His 48; mutants altered at this position are non-chemotactic. Similar mechanisms of transient protein phosphorylation and phosphoryl group transfer seem to be involved in processing sensory data and in activating specific gene expression. SO - Nature 1988 Nov 10;336(6195):139-43. Pos-24. PMID:6292477 | PIR:TVFV60 TI - DNA sequence of the viral and cellular src gene of chickens. 1. Complete nucleotide sequence of an EcoRI fragment of recovered avian sarcoma virus which codes for gp37 and pp60src. AB - Recovered avian sarcoma virus is a class of virus obtained from chicken tumors induced by mutants of Rous sarcoma virus which have a deletion in the src gene. We have determined the entire nucleotide sequence of a 3.1-kilobase EcoRI DNA fragment of molecularly cloned recovered avian sarcoma virus DNA. This DNA fragment contains part of the env gene and the entire src gene. Amino acid sequences of both gene products were deduced from the DNA sequences; the predicted amino acid sequences were verified by protein studies. An env protein (gp37) was found to be composed of 205 amino acids with three glycosylation sites. gp37 had a long stretch of hydrophobic residues near the carboxyl terminus. The src gene product, pp60src, was composed of 526 amino acids and contained the possible sites for tyrosine and serine phosphorylation. The amino acid sequences predicted in this study differ significantly from the amino acid sequence predicted previously for the Schmidt-Ruppin strain of Rous sarcoma virus. SO - J Virol 1982 Oct;44(1):1-11. Pos-25. PMID:8257688 | PIR:IGHUR1 INHUR TI - Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification. AB - Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor. of Hope, Duarte, California 91010. SO - Biochemistry 1993 Dec 14;32(49):13531-6. Pos-26. PMID:2530230 | PIR:MWAXIC TI - The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I. An improved method for locating the phosphorylated amino acid. AB - The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed only when a single site in their heavy chains is phosphorylated by a myosin I heavy chain-specific kinase. We show that phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in myosin IC, and a threonine residue at a corresponding position in myosin IA whose amino acid sequence is as yet unknown. The most obvious feature common to the three substrates is a basic amino acid(s) 2 or 3 residues before the site of phosphorylation. The phosphorylation site is located between the ATP- and actin-binding sites, which corresponds to the middle of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence similarity between the region surrounding the phosphorylation site of myosin I and subfragment 1 is much lower than the average sequence similarity between myosin I and subfragment 1. This is consistent with the hypothesis that the conformation of this region of myosin I differs from that of the corresponding region in skeletal muscle myosin and that phosphorylation converts the conformation of the actomyosin I complex into a conformation comparable to that present in actosubfragment 1 without phosphorylation. The protein sequences obtained in the course of this work led to the conclusion that the myosin I genes previously identified as myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and IB heavy chains, respectively. Finally, we report a modification of the method for monitoring the appearance of 32Pi during sequencing of 32P-labeled peptides that results in almost complete recovery of the radioactivity, thus allowing unequivocal assignment of the position of the phosphorylated residue. Bethesda, Maryland 20892. SO - J Biol Chem 1989 Nov 15;264(32):19340-8. Pos-27. PMID:2210381 | PIR:A38379 JH0216 TI - Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland. AB - A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland. NIH, Bethesda, MD 20892. SO - Gene 1990 Jul 16;91(2):209-15. Pos-28. PMID:2760016 | PIR:JU0038 TI - Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1. AB - The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile. SO - J Biochem (Tokyo) 1989 Apr;105(4):577-81. Pos-29. PMID:8061611 | PIR:GEBOM GEHUM GERTM1 TI - Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and human. AB - The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5. The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides. A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS) 92093-0322. SO - Protein Sci 1994 May;3(5):822-30. Pos-30. PMID:6304091 | PIR:OKBOG TI - Amino acid sequence around a "hinge" region and its "autophosphorylation" site in bovine Lung cGMP-dependent protein kinase. AB - An exposed "hinge" region of cGMP-dependent protein kinase is known to be susceptible to both limited proteolysis and autophosphorylation. A 91-residue fragment has been isolated from this region and its amino acid sequence has been compared with the analogous regions of the cAMP-dependent protein kinases. Although a resemblance among these sequences is not striking, the phosphorylation sites are in corresponding regions toward the NH2 termini, and there are indications of homology in the vicinity of their autophosphorylation sites. As in the cAMP-dependent protein kinase, the site of autophosphorylation and the site of susceptibility to limited proteolysis are very near each other in the primary structure. The actual site of autophosphorylation (the underlined threonine residue in Pro-Arg-Thr-Thr-Arg) is quite different from those in the regulatory subunit of Type II cAMP-dependent kinase or the site in Type I regulatory subunit that can be phosphorylated by the cGMP-dependent protein kinase. SO - J Biol Chem 1983 May 10;258(9):5531-6. Pos-31. PMID:7822248 | PIR:A45100 TI - Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis. AB - Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogen-activated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro. SO - J Biochem (Tokyo) 1994 Aug;116(2):304-14. Pos-32. PMID:2755948 | PIR:S06102 TI - Sequence of caprine alpha s1-casein and characterization of those of its genetic variants which are synthesized at a high level, alpha s1-CnA, B and C. AB - The sequence of caprine alpha s1-casein (199 residues) was established. The peptide chain has the same length as, and shows a 88% degree of identity with, its bovine counterpart. With the ovine alpha s1-casein, the sequence of which was deduced from that of its mRNA, the degree of identity is 97%, counting as one difference a deletion of eight residues in the ovine protein. The differences between the three genetic variants associated with a high alpha s1-casein content in milk are simple substitutions. Variant alpha s1-CnA differs from variant alpha s1-CnB by two substitutions, 16 Leu (A)----Pro (B) and 77 Gln (A)----Glu (B), the latter inducing the appearance of a phosphate group on 75 Ser. Variant alpha s1-CnC differs from alpha s1-CnB by three substitutions, 8 His (B)----Ile (C), 100 Arg (B)----Lys (C) and 195 Thr (B)----Ala (C). The original type of caprine alpha s1-casein could be another hypothetical genetic variant, having the same electrophoretic mobility as alpha s1-CnB. France. SO - Protein Seq Data Anal 1989 Apr;2(3):181-8. Pos-33. PMID:1778990 | PIR:KIRTC2 TI - Phosphorylation of type II (beta) protein kinase C by casein kinase II. AB - Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance. SO - J Biochem (Tokyo) 1991 Oct;110(4):655-60. Pos-34. PMID:1377674 | PIR:DVHUCF TI - Phosphorylation of the cystic fibrosis transmembrane conductance regulator. AB - Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways. New York, New York 10021. SO - J Biol Chem 1992 Jun 25;267(18):12742-52. Pos-35. PMID:8376365 | PIR:A40936 TI - Multiple phosphorylation of stathmin. Identification of four sites phosphorylated in intact cells and in vitro by cyclic AMP-dependent protein kinase and p34cdc2. AB - Stathmin is a ubiquitous, highly conserved phosphoprotein which most likely acts as a relay integrating various intracellular pathways regulating cell proliferation, differentiation, and functions. At least 14 molecular forms of stathmin have been identified so far, which migrate as 2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) = 19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and whose pattern may reflect the state of activation of cells. We found that stathmin could be phosphorylated in vitro by at least three different protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase II, cAMP-dependent protein kinase catalyzed the phosphorylation of stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K). Interestingly, phosphorylation by both kinases together yielded all of the phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide analysis allowed us to demonstrate that the same four sites were exclusively found to be phosphorylated in vivo, in brain tissue as well as in control or nerve growth factor-stimulated PC12 cells. In this latter case, the major site phosphorylated in response to nerve growth factor being serine 25, it is likely that a kinase such as a mitogen-activated protein kinase, known to be activated by growth factors, might directly phosphorylate stathmin. The phosphopeptide map analysis allowed further identification of the specific combinations among the four sites whose phosphorylation is responsible for the characteristic two-dimensional polyacrylamide gel electrophoresis migration of the resulting stathmin forms both in vitro and in vivo and revealed the existence of likely structural interactions between the sites phosphorylated. In conclusion, our results show that phosphorylation of serines 16, 25, 38, and 63 accounts for all of the major functional stathmin forms observed in vivo. The present identification of these sites will foster a better understanding of some intracellular mechanisms involved in the diverse physiological regulation of the proliferation, differentiation, and functions of cells, including the role of stathmin in these processes as a relay integrating diverse signaling pathways. Centre National de la Recherche Scientifique Unite de Recherche Associee 614, Paris, France. SO - J Biol Chem 1993 Sep 25;268(27):20076-84. Pos-36. PMID:7687605 | PIR:A35702 TI - Isolation and characterization of a regulated form of actin depolymerizing factor. AB - Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity. purification/*metabolism SO - J Cell Biol 1993 Aug;122(3):623-33. Pos-37. PMID:6349995 | PIR:HSIN21 TI - Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin. AB - The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation. SO - Eur J Biochem 1983 Sep 1;135(1):113-21. Pos-38. PMID:2164212 | PIR:A31780 TI - Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites. AB - We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities. Dallas, TX 75216. SO - Proc Natl Acad Sci U S A 1990 Jul;87(13):4951-5. Pos-39. PMID:3134350 | PIR:A33369 TI - Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit. AB - Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes. Indianapolis 46223. SO - J Biol Chem 1988 Aug 5;263(22):10561-7. Pos-40. PMID:697844 | PIR:SYRTPP TI - Phosphorylation, a factor controlling the synthesis of L-erythrodihydrobiopterin (BH2). SO - Biochem Biophys Res Commun 1978 Jul 28;83(2):593-8. Pos-41. PMID:6264320 | PIR:TVFV60 TVFVR TI - Homologous tyrosine phosphorylation sites in transformation-specific gene products of distinct avian sarcoma viruses. SO - Nature 1981 Jun 25;291(5817):675-7. Pos-42. PMID:3224509 | PIR:PL0040 TI - The sequence around the phosphorylation site of the porcine heart type phosphorylase isoenzyme. AB - 1. A tetradecapeptide containing the phosphorylation site was obtained from 32P-labelled pig heart phosphorylase a isoenzyme by alpha-chymotryptic digestion. 2. The peptide was purified by Mono S cation-exchange chromatography and reversed-phase HPLC. 3. The phosphorylated residue was identified as Ser and the sequence was determined: T D G E R R K Q I S V R G L. 4. The sequence was compared to the known sequences of muscle and liver type isophosphorylases and the structural consequences of the amino acid residue exchanges were predicted. Hungary. SO - Comp Biochem Physiol B 1988;91(4):717-21. Pos-43. PMID:2532591 | PIR:PL0162 TI - Phosphorylation of paramyosin. AB - 1. Myofibrils isolated from Mercenaria mercenaria were phosphorylated by endogenous kinase. Over a range of ionic strengths only paramyosin was phosphorylated. 2. Thiophosphorylation of paramyosin caused an inhibition of steady-state actin-activated ATPase activity of the myofibrils. 3. It is proposed that the endogenous kinase is the catalytic subunit of the cAMP-dependent protein kinase. 4. The sequence around the phosphorylation site was determined. 5. The phosphorylation site probably is close to the C-terminus of the paramyosin molecule. Tokyo, Japan. SO - Comp Biochem Physiol B 1989;94(4):813-21. Pos-44. PMID:1737801 | PIR:A40936 TI - Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry. Identification of two proline-directed serine phosphorylation sites and a blocked amino terminus. AB - p19 is a highly conserved 19-kDa cytosolic protein that undergoes phosphorylation in mammalian cells upon activation of several distinct signal transduction pathways. Its expression is widespread but developmentally regulated. To determine the in vivo phosphorylation site(s) of p19, the protein was purified from bovine brain and resolved into the unphosphorylated form (p19) and a mixture of the two predominant phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses corresponding to fragments spanning the entire amino acid sequence as deduced from the cDNA except for those predicted to contain an unmodified amino terminus. Instead, the digests revealed ions corresponding to peptides lacking the initiator methionine and containing an N-acetylated alanine at the amino terminus. The analysis of pp19, but not that of p19, revealed two sets of ions representing peptides whose m/z values differed by 80 atomic mass units, the incremental mass of a phosphate residue. These putative phosphate-bearing peptides were sensitive to alkaline phosphatase treatment. Using combined trypsin and V8 protease digestions, the phosphorylation sites were mapped to Ser-25 and Ser-38, in the peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys, respectively. Interestingly, both phosphoserines are in a very similar sequence context, suggesting that a single proline-directed serine protein kinase, possibly p34cdc2, is responsible for phosphorylation of both sites in vivo. New York 10461. SO - J Biol Chem 1992 Feb 15;267(5):3506-13. Pos-45. PMID:8128219 | PIR:TPHUN1 TI - Crystal structure of human protein tyrosine phosphatase 1B. AB - Protein tyrosine phosphatases (PTPs) constitute a family of receptor-like and cytoplasmic signal transducing enzymes that catalyze the dephosphorylation of phosphotyrosine residues and are characterized by homologous catalytic domains. The crystal structure of a representative member of this family, the 37-kilodalton form (residues 1 to 321) of PTP1B, has been determined at 2.8 A resolution. The enzyme consists of a single domain with the catalytic site located at the base of a shallow cleft. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. This site is formed from an 11-residue sequence motif that is diagnostic of PTPs and the dual specificity phosphatases, and that contains the catalytically essential cysteine and arginine residues. The position of the invariant cysteine residue within the phosphate binding site is consistent with its role as a nucleophile in the catalytic reaction. The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation. 11724. purification/metabolism SO - Science 1994 Mar 11;263(5152):1397-404. Pos-46. PMID:2834385 | PIR:KIZMPO TI - Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase. AB - The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence. SO - J Biol Chem 1988 May 15;263(14):6683-7. Pos-47. PMID:8524294 | PIR:T45138 TI - Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant. AB - We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery. SO - Mol Cell Biol 1996 Jan;16(1):179-91. Pos-48. PMID:7997262 | PIR:INHUR TI - Crystal structure of the tyrosine kinase domain of the human insulin receptor. AB - The X-ray crystal structure of the tyrosine kinase domain of the human insulin receptor has been determined by multiwavelength anomalous diffraction phasing and refined to 2.1 A resolution. The structure reveals the determinants of substrate preference for tyrosine rather than serine or threonine and a novel autoinhibition mechanism whereby one of the tyrosines that is autophosphorylated in response to insulin, Tyr 1,162, is bound in the active site. New York, New York 10032. SO - Nature 1994 Dec 22-29;372(6508):746-54. Pos-49. PMID:2108025 | PIR:A31334 A31758 TI - Localization of phosphoserine residues in the alpha subunit of rabbit skeletal muscle phosphorylase kinase. AB - The alpha subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous beta subunit contains all the phosphoserine residues so far identified in the alpha subunit. Federal Republic of Germany. SO - Eur J Biochem 1990 Mar 10;188(2):367-76. Pos-50. PMID:8300603 | PIR:HSBO3 TI - Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a nuclear kinase from mouse leukemia cells. AB - A Ca(2+)-calmodulin dependent histone 3 kinase was partially purified from a low salt (150 mM NaCl) nuclear extract of mouse leukemia cells by calmodulin-Sepharose affinity chromatography. In vitro, the kinase activity transferred gamma-phosphate from ATP to histone 3 to form an acid-labile and alkaline-stable linkage. Under the assay conditions 1.8 mol of phosphate are incorporated per mol of histone 3. Upon modification of arginine residues with phenylglyoxal prior to phosphorylation, a considerable decrease in the amount of phosphate transferred to histone 3 was observed. Amino acid analysis revealed that H3 was phosphorylated on arginine residues. To identify the phosphorylated peptide(s), histone 3 was cleaved with cyanogen bromide prior to phosphorylation. The phosphorylated mixture was then separated by gel filtration high-performance liquid chromatography under denaturing conditions. Fragments I (N-terminal 10.3-kDa peptide) and III (C-terminal 1.7-kDa peptide) were both phosphorylated. Amino acid sequencing further revealed that the molar yields of 3 of the 4 arginines present in the phosphorylated cyanogen bromide fragment III were reduced by a factor of about 10 compared with the corresponding arginines from the unphosphorylated fragment. In the case of fragment I, 25 cycles of Edman degradation revealed that the recovery of only arginine 2 was reduced by a factor of 20. The putative phosphorylation sites are arginines 2, 128, 129, and 131. The sequence information offered an indirect evidence that these arginines were the sites of phosphorylation. The kinase described in this report represents a first member of a potentially important new class of kinases which are Ca(2+)-calmodulin dependent and which phosphorylate arginine. of Chicago, Maywood, Illinois 60153. purification/*metabolism SO - J Biol Chem 1994 Jan 28;269(4):2722-7. Pos-51. PMID:2503376 | PIR:S05207 TI - Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain. Identification of the phosphoserine sites and their influence on filament formation. AB - The in vitro phosphorylation of vimentin, the intermediate filament protein of mesenchymal cells, by kinases A and C is serine-specific and involves only the N-terminal head domain. In oligomeric protofilament units each kinase recognizes five sites, which have been identified by sequence analysis. Kinase C introduces 1.5 mol phosphate/mol vimentin, while kinase A treatment results in 4 mol phosphate/mol. Kinase-A-treated oligomers do not polymerize in standard assays whereas kinase C treatment has no inhibitory effect. Filaments exposed to kinase A remain stable and incorporate only 1.7 mol phosphate/mol vimentin. These phosphates are essentially restricted to two of the five kinase A sites found in protofilament units. Thus the head domain, previously related to in vitro assembly competence and filament stability, changes in accessibility between the oligomeric and polymeric state. We discuss the possibility that in vivo phosphorylation of vimentin filaments by kinase A may not necessarily be accompanied by an extensive depolymerization. It could instead involve a dynamic change of the filament surfaces, which could alter the interaction of the filaments with other cellular structures. Republic of Germany. SO - Eur J Biochem 1989 Aug 1;183(2):441-7. Pos-52. PMID:3680273 | PIR:ACRYD1 TI - Determination of the sites of cAMP-dependent phosphorylation on the nicotinic acetylcholine receptor. AB - The nicotinic acetylcholine receptor is a substrate for cAMP-dependent protein kinase both in vitro and in vivo. Recently, it has been demonstrated that phosphorylation of the nicotinic receptor by this kinase increases its rate of rapid desensitization. We now report the identification of the cAMP-dependent phosphorylation sites on the gamma and delta subunits. Two-dimensional phosphopeptide mapping of the phosphorylated gamma and delta subunits, after limit proteolysis with thermolysin, indicated that each subunit is phosphorylated on a single site. Phosphoamino acid analysis of the 32P-labeled subunits demonstrates that phosphorylation had occurred exclusively on serine residues. Purified phosphorylated subunits were cleaved with cyanogen bromide and the resultant phosphopeptides were purified by reverse-phase high performance liquid chromatography. Shorter phosphopeptides, obtained by secondary digestion with trypsin, were purified and subjected to both automated gas-phase sequencing and manual Edman degradation. The results demonstrate that the gamma subunit was phosphorylated at Ser-353, contained within the sequence Arg-Arg-Ser(P)-Ser-Phe-Ile and that the delta subunit was phosphorylated at Ser-361, contained within the sequence Arg-Ser-Ser(P)-Ser-Val-Gay-Tyr-Ser-Lys. Determination of the sites phosphorylated within the structure of the gamma and delta subunits should contribute to the molecular characterization of the regulation of desensitization of the nicotinic acetylcholine receptor by protein phosphorylation. New York, New York 10021. SO - J Biol Chem 1987 Dec 5;262(34):16748-53. Pos-53. PMID:2019567 | PIR:VEHY TI - The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site. AB - The disassembly of vimentin-containing intermediate filament (IF) networks during mitosis in BHK-21 cells is accompanied by increased phosphorylation of vimentin (Chou, Y.-H., Rosevear, E., and Goldman, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1885-1889). We have recently identified p34cdc2 as the catalytic subunit of one of the two endogenous vimentin kinases in mitotic baby hamster kidney cells (Chou, Y.-H., Bischoff, J. R., Beach, D., and Goldman, R. D. (1990) Cell 62, 1063-1071). To begin to characterize the biochemical basis of the p34cdc2-mediated IF disassembly process, we have purified and sequenced the 32P-labeled tryptic peptides derived from in vitro-phosphorylated vimentin. The results demonstrate that Ser-55, in the N-terminal non-alpha-helical domain of vimentin, is the most favored phosphorylation site. This finding supports the idea that the N-terminal domain of type III IF protein plays a crucial role in regulating IF structure and supramolecular organization. University, Chicago, Illinois 60611. SO - J Biol Chem 1991 Apr 25;266(12):7325-8. Pos-54. PMID:7615564 | PIR:A35702 TI - Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. AB - Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site. University, Fort Collins 80523, USA. SO - J Biol Chem 1995 Jul 21;270(29):17582-7. Pos-55. PMID:2261989 | PIR:S15815 TI - Three phosphorylation sites in elongation factor 2. AB - Elongation factor 2 (EF-2) of rabbit reticulocytes was phosphorylated in vitro by incubation with partially purified EF-2 kinase and [gamma-32P]ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were revealed by two-dimensional peptide mapping. The phosphopeptides were isolated by high performance liquid chromatography and sequenced. A comparison of the primary structure of the phosphopeptides with that of EF-2 showed that all 4 phosphopeptides originated from one region of EF-2 located near the N-terminus that contains 3 threonine residues: Thr-53, Thr-56, Thr-58. A direct estimation of localization of radioactive phosphate in the phosphopeptides demonstrated that all the enumerated threonine residues in EF-2 can be phosphorylated in vitro. Moscow Region. SO - FEBS Lett 1990 Nov 26;275(1-2):209-12. Pos-56. PMID:8226879 | PIR:QRBOT1 QRBOT2 TI - Brain proline-directed protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments. AB - Brain proline-directed protein kinase (BPDK), which contains a catalytic subunit homologous to and displaying site-specific phosphorylation similar to p34cdc2 kinase (Lew, J., Winkfein, R. J., Paudel, H. K., and Wang, J. H. (1992) J. Biol. Chem. 267, 25922-25926), has been examined for possible involvement in tau phosphorylation. Immunoblot analyses using peptide antibodies specific for BPDK have revealed the presence of the kinase in bovine brain microtubules purified extensively by repeated polymerization and depolymerization cycles. When the microtubule proteins are separated into the tubulin and microtubule-associated protein fractions, BPDK is found exclusively in the latter fraction. BPDK phosphorylates both tau and MAP2, the former protein being phosphorylated to a stoichiometry of 3.8 mol of phosphate/mol of tau. Analysis of the phosphopeptides isolated from the tryptic digest of the phosphorylated bovine tau has revealed seven phosphorylation sites. Based on the sequence alignment between bovine and human tau proteins, these sites correspond to Ser-195, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, and Ser-404 of human tau. Mass spectrometric analysis of the tau protein isolated from Alzheimer's paired helical filaments (PHFs) has determined three abnormal phosphorylation sites and two phosphopeptides containing a total of five abnormal phosphates (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 267, 17047-17054). Two of the sites in tau phosphorylated by BPDK, Thr-231 and Ser-235, are among the abnormal phosphorylation sites, and the other sites phosphorylated by BPDK are within phosphopeptides from PHF-tau. These results suggest that BPDK may be one of the kinases responsible for the abnormal phosphorylation-associated PHF-tau. Calgary, Alberta, Canada. SO - J Biol Chem 1993 Nov 5;268(31):23512-8. Pos-57. PMID:838735 | PIR:SBHUP TI - Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. AB - The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted. SO - J Biol Chem 1977 Mar 10;252(5):1689-95. Pos-58. PMID:2756156 | PIR:GMDG GMPGB TI - The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum. AB - Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms. SO - Regul Pept 1989 May;25(2):223-33. Pos-59. PMID:3928373 | PIR:A33369 TI - Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I. AB - A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated. SO - Eur J Biochem 1985 Aug 15;151(1):39-48. Pos-60. PMID:8635594 | PIR:C1HURB TI - Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction. AB - Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule. Jean-Pierre Ebel, Grenoble, France. SO - FEBS Lett 1996 May 13;386(1):15-20. Pos-61. PMID:2544997 | PIR:INHUR TI - Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor. AB - The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes. Medicine, Inohana, Japan. SO - Science 1989 Jul 7;245(4913):63-6. Pos-62. PMID:2211643 | PIR:A40936 TI - Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes. AB - Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells. SO - J Biol Chem 1990 Oct 15;265(29):17499-505. Pos-63. PMID:2912504 | PIR:PZRB1 TI - Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues. AB - Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue. SO - Biochim Biophys Acta 1989 Feb 9;1010(2):218-26. Pos-64. PMID:2500966 | PIR:A43803 TI - Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure. AB - We reported that stoichiometric phosphorylation by either cAMP-dependent protein kinase or protein kinase C induces disassembly of vimentin filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato, C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263, 5970-5978]. In the present work, we attempted to identify the sites of vimentin phosphorylated by each protein kinase. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were specifically phosphorylated by protein kinase C, whereas Ser-46 was phosphorylated preferentially by cAMP-dependent protein kinase. Both kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific phosphorylation sites for protein kinase C are mostly located close to the amino-terminal side of arginine while those for cAMP-dependent protein kinase are located close to the carboxyl-terminal side of arginine. The phosphorylation sites exclusively occur in the amino-terminal non-alpha-helical head domain, particularly at the beta-turn region. These results provide clues to the molecular mechanisms of phosphorylation-dependent disassembly of vimentin filaments. SO - Biochemistry 1989 Apr 4;28(7):2974-9. Pos-65. PMID:2721673 | PIR:UDCH TI - The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein. AB - Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion-exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1. SO - FEBS Lett 1989 May 8;248(1-2):162-8. Pos-66. PMID:7909431 | PIR:DVHU1 TI - Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. AB - Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule. GA 30322. SO - Biochem J 1994 Apr 1;299 ( Pt 1):309-15. Pos-67. PMID:1696913 | PIR:SGHU1V TI - The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent. AB - In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function. Israel. SO - FEBS Lett 1990 Aug 20;269(1):221-5. Pos-68. PMID:3137939 | PIR:A33369 TI - Phosphorylation of glycogen synthase by a bovine thymus protein-tyrosine kinase, p40. AB - Glycogen synthase from rabbit skeletal muscle was found to be phosphorylated by a protein-tyrosine kinase, p40, purified from bovine thymus. The phosphorylation, to a stoichiometry of 0.4-0.5 mol/mol subunit, was specific for a single tyrosine residue in the sequence EEDGERYDEDEE. This acidic sequence has considerable similarity to the site recognized by p40 in erythrocyte band 3 protein. In the analysis of the phosphorylated peptide, it was noted that the sequence -RY(P)- impeded cleavage by either trypsin or automatic Edman degradation. Indianapolis 46223. SO - Biochem Biophys Res Commun 1988 Aug 30;155(1):52-8. Pos-69. PMID:1869528 | PIR:S57631 TI - A major substrate of maturation promoting factor identified as elongation factor 1 beta gamma delta in Xenopus laevis. AB - Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Belle, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis. of Leiden, The Netherlands. SO - J Biol Chem 1991 Aug 15;266(23):14885-8. Pos-70. PMID:8654396 | PIR:EQBOA TI - The C-terminal bisphosphorylated proenkephalin-A-(209-237)-peptide from adrenal medullary chromaffin granules possesses antibacterial activity. AB - The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C-terminal chromogranin B-derived peptide 614-626 which was shown to have antibacterial activity [Strub, J.M., Garcia-Sablone, P., Looning, K., Taupenot, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz-Boutigue, M.H. (1995) Eur. J. Biochem. 229, 356-368]. We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C-terminal 209-237 sequence of proenkephalin-A. A detailed study using microsequencing and matrix-assisted-laser-desorption time-of-flight mass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenous bisphosphorylated proenkephalin-A-(209-237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2 - 0.4 microM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C-terminal domain of diazepam-binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relationship to its biological activity. Biologie de la Communication Cellulaire, Strasbourg, France. SO - Eur J Biochem 1996 Feb 1;235(3):516-25. Pos-71. PMID:2448300 | PIR:SGHU1V TI - Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain. AB - Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis. St. Louis, Missouri 63110. SO - J Biol Chem 1988 Feb 5;263(4):1942-5. Pos-72. PMID:3200826 | PIR:A31758 TI - The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit. AB - We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains. Republic of Germany. SO - Proc Natl Acad Sci U S A 1988 Dec;85(24):9381-5. Pos-73. PMID:2106518 | PIR:R3RTS6 TI - Hormonally inducible phosphorylation of a nuclear pool of ribosomal protein S6. AB - Thyrotropin releasing hormone (TRH) and epidermal growth factor induce the rapid phosphorylation of a basic, chromatin-associated protein present in GH4 rat pituitary cells and also found in primary hepatocyte culture. Cell fraction experiments indicate a nucleolar localization for this basic, chromatin-associated protein. The protein has been purified to homogeneity from rat liver and 23 amino acids of its N-terminal sequence determined. There is complete homology between the sequenced portion of the basic, chromatin-associated protein and the N-terminal sequence of rat ribosomal protein S6. In vivo and in vitro phosphorylation, two-dimensional gel analysis and two-dimensional tryptic phosphopeptide mapping support the identity of the basic, chromatin-associated protein and S6. Our experimental data indicate the existence of a nuclear pool of S6 whose phosphorylation is hormone inducible. School of Medicine, La Jolla 92093. SO - J Biol Chem 1990 Mar 15;265(8):4321-5. Pos-74. PMID:3166375 | PIR:INHUR TI - Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping. AB - 1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells. Bristol, U.K. SO - Biochem J 1988 Jun 1;252(2):607-15. Pos-75. PMID:2394752 | PIR:A38379 TI - Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. AB - Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than 98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo. Angeles 90024. SO - J Biol Chem 1990 Sep 15;265(26):15860-6. Pos-76. PMID:2109669 | PIR:A60521 TI - Purification and characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada: amino acid sequence of the phosphorylation site. AB - 1. Skeletal muscle glycogen phosphorylase b has been purified from Liza ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be 97,000. By gel filtration a relative Mr of 190,000 was found. 3. Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a [32P]-labelled peptide which is observed to contain Ser, its sequence being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of mullet phosphorylase, 32P was incorporated in different protein bands resolved by isoelectric focusing. The degree of radioactivity associated with each one changed with the incubation time. Spain. SO - Comp Biochem Physiol B 1990;95(2):295-301. Pos-77. PMID:3514596 | PIR:PEMQAJ TI - The complete amino acid sequence of monkey pepsinogen A. AB - The complete amino acid sequence of pepsinogen A from the Japanese monkey (Macaca fuscata) was determined. After converting the pepsinogen to pepsin by activation, the pepsin moiety was reduced and carboxymethylated, cleaved by cyanogen bromide, and the amino acid sequences of the major fragments determined. These fragments were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of intact pepsin. Since the sequence of the activation segment had been determined previously (Kageyama, T., and Takahashi, K. (1980) J. Biochem. (Tokyo) 88, 9-16), the 373-residue sequence of monkey pepsinogen A was established, consisting of the pepsin moiety of 326 residues and the activation segment of 47 residues. Three disulfide bridges and 1 phosphoserine residue were found to be present in the pepsinogen molecule. The molecular weight was calculated to be 40,027 including the phosphate group. Monkey pepsinogen A showed high homology with human (94% identity) and porcine (86% identity) pepsinogens A. SO - J Biol Chem 1986 Apr 5;261(10):4395-405. Pos-78. PMID:6993480 | PIR:TMRBA TI - The amino acid sequence of rabbit cardiac tropomyosin. AB - The amino acid sequence of rabbit cardiac tropomyosin has been investigated by the isolation of peptides derives from cyanogen bromide and proteolytic degradations. Based on identical amino acid compositions, electrophoretic mobilities, and in some cases, NH2-terminal analyses, all peptides were shown to be the same as peptides in the amino acid sequence of rabbit skeletal alpha-tropomyosin. The apparent heterogeneity of the cardiac protein previously observed on alkaline-urea polyacrylamide electrophoresis gels is attributable to a phosphorylated species. We conclude that the primary structures of the cardiac and skeletal alpha component are identical. Differences in actin-linked calcium regulation in skeletal and cardiac muscle must reside largely in the troponin components. SO - J Biol Chem 1980 Jul 25;255(14):6854-9. Pos-79. PMID:2346743 | PIR:A34594 TI - Phosphorylation of bovine platelet myosin by protein kinase C. AB - Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation. Cleveland, Ohio 44106. SO - Biochemistry 1990 Mar 20;29(11):2713-20. Pos-80. PMID:2501795 | PIR:QRBOT1 TI - Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. AB - Amino acid sequencing of a CNBr digest of the tau protein isolated from bovine brain revealed an amino acid sequence of 17 residues, Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, which we call peptide I, with heterogeneity at position 11 of glycine (peptide Ia) and proline (peptide Ib); peptide I showed no homology with the previously reported cDNA-derived mouse and human tau sequences. Antisera raised to synthetic peptides corresponding to peptides Ia and Ib labeled all the bovine tau polypeptides recognized by other monoclonal and polyclonal antibodies to bovine tau. Antisera to peptide Ib did not label any mouse tau polypeptides; however, an anti-Ia antiserum labeled two of the four mouse tau polypeptides. Antisera to both peptides labeled paired helical filaments (PHF) as neurofibrillary tangles, plaque neurites, and neuropil threads in Alzheimer disease brain and PHF polypeptides on immunoblots. Immunostaining with anti-Ia antisera of PHF in tissue sections and PHF polypeptides, but not bovine tau, on immunoblots was markedly increased when pretreated with alkaline phosphatase. These studies suggest that (i) the amino acid sequences of some isoforms of tau peptide might be different from that predicted from cDNAs, (ii) a tau peptide that is absent in the predicted sequences is present in PHF in Alzheimer disease, and (iii) tau in PHF is abnormally phosphorylated. Staten Island 10314. SO - Proc Natl Acad Sci U S A 1989 Jul;86(14):5646-50. Pos-81. PMID:2914943 | PIR:EQBOA TI - The isolation and chemical characterization of phosphorylated enkephalin-containing peptides from bovine adrenal medulla. AB - There is increasing evidence that the opioid peptide precursor, proenkephalin A, and its products undergo extensive post-translational modification, in addition to the cleavage at dibasic amino acid sites. We have used an antiserum directed toward the C terminus of Met-enkephalin Arg6-Phe7 in a radioimmunoassay to monitor the purification to homogeneity of four peptide B variants from bovine adrenal medulla, using gel filtration, anion exchange chromatography, and reverse phase high performance liquid chromatography. Amino acid sequence analysis, together with immunochemical data, confirmed that each comprised the primary sequence, proenkephalin A-(209-239). In addition, three of the four variants were shown to be phosphorylated by alkaline phosphatase digestion, microphosphate analysis, and ethanethiol derivatization coupled with amino acid sequence analysis; these variants were shown to have 1, 2, or 3 phosphate groups per peptide chain, which corresponded to their increasing acidic nature. The phosphorylation sites were clustered together at positions Ser7, Ser13, and Ser15 and were in close association with acidic residues. The clustering of phosphorylated residues is unique among regulatory peptide precursors. This region of proenkephalin A is well conserved, which suggests that it constitutes an important novel functional domain. Liverpool, United Kingdom. purification/metabolism SO - J Biol Chem 1989 Feb 25;264(6):3061-5. Pos-82. PMID:1939059 | PIR:A33430 TI - Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites. AB - It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin. SO - J Biol Chem 1991 Oct 25;266(30):19971-5. Pos-83. PMID:2211670 | PIR:A28822 TI - Feedback regulation of phospholipase C-beta by protein kinase C. AB - Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation. National Institutes of Health, Bethesda, Maryland 20892. SO - J Biol Chem 1990 Oct 15;265(29):17941-5. Pos-84. PMID:3121625 | PIR:F2SP44 F2SPD2 FMSP32 TI - Tandem mass spectrometry reveals that three photosystem II proteins of spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2 termini. AB - Photosystem II cores of spinach contain four phosphoproteins (8.3, 32, 34, and 44 kDa). Tryptic digestion of core particles released four phosphopeptides which were purified by affinity chromatography on Fe3+-chelating Sepharose and reverse-phase high pressure liquid chromatography. One peptide, derived from the 8.3-kDa protein, has been found to be the NH2 terminus of the psbH gene product (Michel, H. P., and Bennett, J. (1987) FEBS Lett. 212, 103-108). The other three peptides were found to be blocked at the NH2 terminus. We now report the use of tandem mass spectrometry to obtain the sequence of the three other peptides, to locate the phosphorylated residue, and to identify the blocking group. The three peptides correspond to the NH2 termini of D1, D2, and CPa-2; and each begins with N-acetyl-O-phosphothreonine. Comparison with sequences deduced from cloned genes indicates that D1 and D2 have lost their initiating N-formylmethionyl residues. The result for D1 contradicts the view that translation of D1 begins at the second AUG of the mRNA (Bloom, M., Brot, N., Cohen, B. N., and Weissbach, H. (1986) Methods Enzymol. 118, 309-315) and supports the view that processing of pre-D1 to its mature form involves loss of amino acids from the COOH terminus (Marder, J. B., Goloubinoff, P., and Edelman, M. (1984) J. Biol. Chem. 259, 3900-3908). In contrast, CPa-2 is processed at the NH2 terminus by cleaving off the first 14 amino acids. These results also establish that the NH2 termini of D1, D2, and CPa-2 are exposed to the stromal side of the thylakoids. SO - J Biol Chem 1988 Jan 25;263(3):1123-30. Pos-85. PMID:2558715 | PIR:A56968 TI - Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain. AB - The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported (Hashimoto et al., 1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released [32P]phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN (Kincaid et al., 1988) allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding site. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM. Alcohol Abuse and Alcoholism, Rockville, Maryland 20852. SO - Biochemistry 1989 Nov 28;28(24):9243-7. Pos-86. PMID:3198618 | PIR:A34594 TI - Sites phosphorylated in myosin light chain in contracting smooth muscle. AB - Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation. Dallas 75235. SO - J Biol Chem 1988 Dec 15;263(35):19166-73. Pos-87. PMID:2037042 | PIR:S15815 TI - Identification of the phosphorylation sites in elongation factor-2 from rabbit reticulocytes. AB - The sites in eukaryotic elongation factor eEF-2 phosphorylated by the Ca2+/calmodulin-dependent eEF-2 kinase in vitro have been identified. The kinase catalysed the rapid incorporation of one mol of phosphate per mol eEF-2 and the slower incorporation of a second mol. All the phosphorylation sites in eEF-2 are contained in the CNBr fragment corresponding to residues 22-155. Tryptic digestion of phosphorylated eEF-2 yielded 3 phosphopeptides, one being unique to monophosphorylated eEF-2. The phosphorylation sites were identified as threonine residues 56 and 58, the former being more rapidly phosphorylated. Ala-Gly-Glu-Thr-Phe-Thr56-Asp-Thr58-Arg. The same sites are labelled in eEF-2 isolated from reticulocyte lysates. Bristol, UK. SO - FEBS Lett 1991 May 6;282(2):253-8. Pos-88. PMID:2842154 | PIR:A33369 TI - Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry. AB - The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo. SO - Eur J Biochem 1988 Aug 15;175(3):497-510. Pos-89. PMID:1281467 | PIR:A45100 TI - Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases. AB - Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase. SO - J Biol Chem 1992 Dec 25;267(36):25628-31. Pos-90. PMID:1835085 | PIR:IQECDK TI - DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. AB - DnaK, the sole Escherichia coli member of the highly conserved 70-kDa heat shock protein (HSP70) family of proteins, autophosphorylates when incubated with ATP in vitro. We show that threonine-199 is the amino acid that becomes phosphorylated and we demonstrate that threonine-199 is critical for the ATPase activity of DnaK. We also report that both the ATPase and autophosphorylating activities of DnaK increase very strongly over the range of temperatures that is physiologically relevant for E. coli growth. The temperature dependence of either or both of these activities could be of significance with respect to the postulated role of DnaK as a molecular chaperone in helping cells ameliorate the deleterious consequences of elevated temperature. Furthermore, we postulate that DnaK plays a key role in regulation of the heat shock response by serving as a cellular thermometer that directly senses the environmental temperature. 02139. SO - Proc Natl Acad Sci U S A 1991 Nov 1;88(21):9513-7. Pos-91. PMID:405007 | PIR:A33369 TI - Amino acid sequence of a phosphorylation site in skeletal muscle glycogen synthetase. SO - Biochem Biophys Res Commun 1977 Apr 11;75(3):643-50. Pos-92. PMID:8639521 | PIR:QRECCS TI - Structure and dynamics of a CheY-binding domain of the chemotaxis kinase CheA determined by nuclear magnetic resonance spectroscopy. AB - The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals. Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which binds to the downstream targets of phosphorylation, the response regulators CheY and CheB. This protein fragment contains the CheY-binding domain flanked on each side by regions that correspond to domain linkers in the intact protein. The structure of the domain was determined from 1429 restraints derived from heteronuclear multidimensional NMR experiments. Hybrid distance geometry--dynamical simulated annealing methods were used to calculate a family of structures that satisfy the experimental distance restraints and torsion angle restraints. The root mean square deviation of the 69 ordered residues in the domain is 0.52 A for the backbone heavy atoms and 0.99 A for all heavy atoms. The residues that have been implicated as important for CheY binding form a face consisting of several partially buried hydrophobic residues, framed by charged residues. The dynamic properties of this protein fragment were measured and analyzed using both isotropic and anisotropic models of molecular motion. The linker regions are very flexible and disordered, as evidenced by the very dynamics properties as compared to the CheY-binding domain. The CheY-binding domain of CheA is structurally similar to the histidine-containing phosphocarrier, HPr, which is a protein involved in the phosphoenolpyruvate:sugar phosphotransferase (PTS) pathway. This structural similarity suggests a possible evolutionary relationship of the PTS and chemotaxis pathways. SO - Biochemistry 1996 May 7;35(18):5633-40. Pos-93. PMID:1869568 | PIR:QRECCY TI - Crystal structure of Escherichia coli CheY refined at 1.7-A resolution. AB - The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57. 60612. SO - J Biol Chem 1991 Aug 15;266(23):15511-9. Pos-94. PMID:2552036 | PIR:A34957 TI - ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Amino acid sequence of ARPP-21B from bovine caudate nucleus. AB - ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS-PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of the rat CNS. It has recently been purified to homogeneity from bovine caudate nucleus and characterized (Hemmings and Greengard, 1989). ARPP-21 is isolated as 2 isoforms, ARPP-21A and ARPP-21B. The amino acid sequence of purified bovine ARPP-21B has now been determined by gas-phase sequencing. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage with trypsin, chymotrypsin, subtilisin, and endoproteinase Lys-C. The resulting peptides were purified by high-performance liquid chromatography, and selected peptides were subjected to amino acid analysis and/or amino acid sequencing by automated Edman degradation. ARPP-21B consists of a single NH2-terminal blocked polypeptide chain of 88 residues, with a calculated molecular mass of 9561 Da, including an NH2-terminal acetyl group inferred by deblocking with an acylaminopeptidase. This molecular mass is significantly lower than earlier estimates based on SDS-PAGE or hydrodynamic measurements. The seryl residue phosphorylated by cAMP-dependent protein kinase (Hemmings et al., 1989) is located at position 55. The molecule contains 1 cysteinyl residue, at position 71, and contains no methionyl, tyrosyl, phenylalanyl, tryptophanyl, or histidinyl residues. Determination of the primary structure of ARPP-21, one of several phosphoproteins localized to dopaminoceptive neurons in the basal ganglia, provides a framework for further investigations into the molecular mechanisms involved in dopaminergic neurotransmission. Haven, Connecticut 06510. SO - J Neurosci 1989 Oct;9(10):3631-7. Pos-95. PMID:2874140 | PIR:WHRTY TI - Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase. AB - As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339], tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated at an identical site (site A) by cyclic AMP-dependent protein kinase, the calmodulin-dependent multiprotein kinase and protein kinase C, while the calmodulin-dependent multiprotein kinase also phosphorylates another unique site (site C). Preparations of tyrosine hydroxylase purified from this source are also contaminated with traces of a fourth protein kinase which phosphorylates another unique site (site E). We have isolated tryptic peptides containing each of these sites and determined their amino acid sequences. By comparison of these data with the known cDNA sequence for rat tyrosine hydroxylase, we have been able to identify these sites as Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also phosphorylated a secondary site which was identified as ser-153. All of these phosphorylation sites are in the amino-terminal region, where there is no significant homology with the closely related enzyme, phenylalanine hydroxylase. Our data also establish that the initiator methionine is removed by post-translational processing to leave pro-2 as the amino-terminus of the mature protein. The significance of these results for the mechanism of action of extracellular signals on catecholamine biosynthesis is discussed. SO - J Biol Chem 1986 Aug 15;261(23):10489-92. Pos-96. PMID:8075074 | PIR:QRECCY TI - Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy. AB - NMR spectroscopy has been used to study recombinant Escherichia coli CheY, a 128-residue protein involved in regulating bacterial chemotaxis. Heteronuclear three- and four-dimensional (3D and 4D) experiments have provided sequence-specific resonance assignments and quantitation of short-, medium-, and long-range distance restraints from nuclear Overhauser enhancement (NOE) intensities. These distance restraints were further supplemented with measurements of three-bond scalar coupling constants to define the local dihedral angles, and with the identification of amide protons undergoing slow solvent exchange from which hydrogen-bonding patterns were identified. The current model structure shows the same global fold of CheY as existing X-ray structures (Volz & Matsumura, 1991; Stock et al. 1993) with a (beta/alpha)5 motif of five parallel beta-strands at the central core surrounded by three alpha-helices on one face and with two on the opposite side. Heteronuclear 15N-1H relaxation experiments are interpreted to show portions of the protein structure in the Mg2+ binding loop are ill-defined because of slow motion (chemical exchange) on the NMR time scale. Moreover, the presence of Mg2+ disrupts the salt bridge between the highly conserved Lys-109 and Asp-57, the site of phosphorylation. SO - Biochemistry 1994 Sep 6;33(35):10731-42. Pos-97. PMID:164350 | PIR:A31334 A31758 TI - The hormonal control of activity of skeletal muscle phosphorylase kinase. Amino-acid sequences at the two sites of action of adenosine-3':5'-monophosphate-dependent protein kinase. AB - Two tryptic phosphopeptides containing the sites on the alpha and beta subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3':5'-monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined. 32P-labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20 degrees C. This treatment released nearly all the 32P radioactivity associated with the beta subunit as trichloroacetic-acid-soluble material. Only a small proportion of the 32P radioactivity associated with the alpha subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet. The beta-subunit tryptic phosphopeptide was completely resolved from traces of the alpha-subunit phosphopeptide by gel filtration on Sephadex G-25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine-amino-acid segment of the betachain, which was found to possess the sequence: Gln-Ser-Gly-Ser(P)-Val-Ile-Tyr-Pro-Leu-Lys. The four components were produced by the partial cyclisation of the N-terminal glutaminyl residue, and by the presence of two alleles for the beta subunit in the rabbit population, which led to a valine-isoleucine ambiguity. The alpha-subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5% trichloroacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G-50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino-terminal sequence of the peptide was shown to be: Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly. The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclic-AMP-dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic-AMP-dependent protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed. SO - Eur J Biochem 1975 Feb 3;51(1):79-92. Pos-98. PMID:6311252 | PIR:OKBO2C TI - Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase. AB - The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein. SO - Biochemistry 1983 Jul 19;22(15):3702-9. Pos-99. PMID:8132603 | PIR:MMECZB TI - Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli. AB - EnvZ is a membrane-bound histidine kinase that functions as an osmotic sensor capable of phosphorylating the regulator protein OmpR in Escherichia coli. To characterize the site of phosphorylation biochemically, we overexpressed a 36-kDa truncated EnvZ protein (Glu-106 to Gly-450) that formed inclusion bodies in the cell. After solubilization, the inclusion body form of EnvZ was cleaved into two major fragments with molecular weights of 25,000 and 10,000. The 25-kDa fragment, EnvZc, was purified and found to exist as a dimer. N-terminal sequence analysis established that cleavage had occurred at Arg-214, indicating that EnvZc contained most of the cytoplasmic domain of EnvZ. After labeling EnvZc with [gamma-32P]ATP, the protein was proteolytically digested, and the resulting peptides were separated by reverse phase chromatography using high performance liquid chromatography. One major radioactive peptide containing greater than 90% of the recovered peptide-associated radioactivity was isolated. Amino acid analysis of this purified peptide indicated that the composition was consistent with a peptide that contained His-243. The amino acid sequence of this peptide was determined to be MAGVSHDLRTP (residues 238-248). These results indicate that His-243 is the major site of phosphorylation on EnvZ and represents the first biochemical characterization of the site of phosphorylation of a membrane histidine kinase of the two-component regulatory family of molecules in bacteria. SO - J Biol Chem 1994 Mar 25;269(12):8728-33. Pos-100. PMID:3087361 | PIR:A33369 TI - Identification of the C-terminus of rabbit skeletal muscle glycogen synthase. AB - The primary structure of a tryptic peptide containing one of the phosphorylation sites on rabbit skeletal muscle glycogen synthase (site 1b) has been redetermined and shown to correspond to the C-terminus of the protein. The sequence is: -SNSVDTSSLSTPSEPLSSAPSLGEERN. SO - Biochem Biophys Res Commun 1986 May 29;137(1):542-5. Pos-101. PMID:3944083 | PIR:A32541 TI - The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion. AB - The neutral histidine-rich polypeptide (HRP) from human parotid secretion was isolated by ion-exchange and gel-filtration chromatography. The complete amino acid sequence determined by automated Edman degradation of the protein, tryptic and Staphylococcus aureus V8 protease peptides, and digestion with carboxypeptidase A is: (Formula: see text) where Pse represents phosphoserine. The polypeptide contains 38 residues and has Mr 4929. The charged amino acids predominate with 7 histidine, 4 arginine, 3 lysine, 3 aspartic acid, 3 glutamic acid residues, and 1 phosphoserine. Assuming minimal charge contributions from histidine and one negative charge from phosphoserine at pH 7, the net charge of HRP is balanced by an equal contribution of basic and acidic residues. Furthermore, the distribution of hydrophilic and hydrophobic residues along the polypeptide chain indicates that there is no structural polarity. The polypeptide lacks threonine, alanine, valine, cysteine, methionine, and isoleucine. HRP did not display sequence similarity with any protein sequence in the National Biomedical Research Foundation Data Bank. HRP is an active inhibitor of hydroxyapatite crystal growth from solutions supersaturated with respect to calcium phosphate salts and therefore must play a role in the stabilization of mineral-solute interactions in oral fluid. In addition, HRP is a potent inhibitor of Candida albicans germination and therefore may be a significant component of the antimicrobial host defense system in the oral cavity. SO - J Biol Chem 1986 Jan 25;261(3):1177-82. Pos-102. PMID:2171679 | PIR:MMHUE4 TI - Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1. AB - In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem. 260, 9073-9076). The 10 kDa domain also contains the spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase inhibits the binding of protein 4.1 to spectrin and actin (Ling, E., Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In this study, we have identified two sites on protein 4.1 which account for 80% of the phosphate incorporated into protein 4.1 during metabolic labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More than 95% of the 32P incorporated into protein 4.1 was in the form of phosphoserine. Reverse-phase HPLC of the peptides generated by digestion of the isolated protein with trypsin or endoproteinase lysine C produced two major radioactive peaks. The phosphorylation sites, identified by gas phase sequencing of the purified phosphopeptides and confirmed by determining the residues converted to S-ethylcysteine by reacting the phosphopeptides with ethanethiol under alkaline conditions, were Ser-331, in the 16 kDa domain and Ser-467, in the 10 kDa domain. 06510. SO - Biochim Biophys Acta 1990 Oct 15;1055(1):87-92. Pos-103. PMID:3345839 | PIR:S00347 TI - Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase. AB - The primary structure of a region on hormone-sensitive lipase was determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser- Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Ly s. Ser-8 was the only residue in the intact protein phosphorylated by cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo. England. SO - FEBS Lett 1988 Feb 29;229(1):68-72. Pos-104. PMID:4369337 | PIR:TPRBIS TI - The phosphorylation sites of troponin I from white skeletal muscle of the rabbit. SO - FEBS Lett 1974 Jun 15;42(3):253-6. Pos-105. PMID:8218377 | PIR:HSHUP1 HSHUP2 TI - Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites. AB - Human sperm is characterized by a high heterogeneity of its basic nuclear protein complement of pro-protamines, protamines and histones. This heterogeneity is increased by the persistence of phosphorylated protamines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to various degrees. Presence of non-phosphorylated and phosphorylated protamines HP1 and HP2 was further demonstrated by electrospray mass spectrometry. Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were identified by automatic Edman degradation of the protein after phosphoserine derivatization to S-ethylcysteine. In both phosphorylated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer electrophoresis. SO - Biochim Biophys Acta 1993 Nov 10;1203(1):109-14. Pos-106. PMID:6318767 | PIR:FGHUA TI - Phosphorylation of fibrinogen by casein kinase 1. AB - Casein kinase 1 phosphorylated human fibrinogen, in a reaction that did not use GTP as phosphoryl donor and was neither stimulated by cyclic AMP or Ca2+, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. Maximal incorporation averaged 4 mol of phosphate per mol of fibrinogen, most of it in the largest CNBr-fragment of the alpha-chain. Phosphoamino acid analysis revealed that phosphorylation occurred only at seryl residues. The phosphorylation of fibrinogen by casein kinase 1 was reverted by alkaline phosphatase. SO - Biochem Biophys Res Commun 1983 Dec 16;117(2):631-6. Pos-107. PMID:7622625 | PIR:TMRBA TI - Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions. AB - When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin. Germany. SO - J Muscle Res Cell Motil 1995 Apr;16(2):103-10. Pos-108. PMID:7107568 | PIR:SBMQPI TI - Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide. AB - Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from pooled human collections displayed the same groups of proteins when fractionated by anion exchange and gel filtration chromatography. We have isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a proline-rich phosphopeptide (M-statherin) from macaque parotid saliva. MPRP has an apparent molecular weight of 16,900 and displays an unusual chemical composition. It is enriched in proline, glycine, and acidic amino acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25% (w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of protein. M-statherin is a 42-residue phosphopeptide with a high proline, glutamic acid, and tyrosine content, but which lacks threonine, valine, cysteine, methionine, isoleucine, and histidine. The complete covalent structure of M-statherin (Mr = 5,368) is: NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Ar g-Tyr- -Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-T yr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of a component in the salivary secretion of a subhuman primate. Phosphoserine occurs at residues 2 and 3. All 13 acidic and basic amino acids are located in the NH2-terminal half of the molecule. The carboxyl-terminal half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is repeated three times, the dipeptide Gln-Pro occurs twice, and the tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once. Evaluation of secondary structure by the Chou-Fasman method predicts an alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the molecule. Both MPRP and M-statherin inhibit spontaneous and seeded precipitation from solutions supersaturated with respect to calcium phosphate salts. This suggests that these macaque compounds may function by maintaining saliva supersaturated with respect to calcium phosphate salts, a necessary requirement for stabilization of hydroxyapatite in the surface layers of teeth.U SO - J Biol Chem 1982 Aug 25;257(16):9271-82. Pos-109. PMID:4369265 | PIR:TPRBIS TI - The amino acid sequences of the phosphorylated sites in troponin-I from rabbit skeletal muscle. SO - FEBS Lett 1974 Jun 15;42(3):249-52. Pos-110. PMID:7783627 | PIR:IQECDK R3EC1 SYECSA TI - Identification of phosphoproteins in Escherichia coli. AB - The substrates of ion- and lipid-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80 kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65 kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium-stimulated protein of 70 kDa was identified as the heat-shock protein DnaK, and a 17 kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase. SO - Mol Microbiol 1995 Feb;15(3):573-80. Neg-1. PMID:2118164 | PIR:F70584 TI - Evidence that protein antigen b of Mycobacterium tuberculosis is involved in phosphate metabolism. AB - Protein antigen b (Pab) of Mycobacterium tuberculosis has previously attracted interest because of its immunological and diagnostic relevance. In this study we present evidence that Pab possesses a signal sequence and is secreted from the cytoplasm of M. tuberculosis. The synthesis of Pab is enhanced under phosphate starvation indicating that the protein is involved in phosphate metabolism in M. tuberculosis. SO - J Gen Microbiol 1990 Mar;136 ( Pt 3):477-80. Neg-2. PMID:3840433 | PIR:A27335 PHRBG TI - Comparative sequence analysis of rat, rabbit, and human muscle glycogen phosphorylase cDNAs. AB - As an initial step in the investigation of the structure, evolution and developmental regulation of the glycogen phosphorylase gene family, we have isolated partial cDNAs to rat, rabbit and human muscle phosphorylase mRNAs. Sequence comparisons of these cDNAs in regions that encode portions of the enzyme located near and encompassing the C terminus show that there is a high degree of interspecies conservation of structure in this region. Conservation of amino acid and nucleotide sequence is high, approximately 96% and 90% homology, respectively, among all three species. In addition, most of the amino acid changes that have occurred conserve the chemical nature of the amino acid side-chains affected. The changes can be easily accommodated in the rabbit muscle phosphorylase tertiary structure and appear to have little effect on the overall conformation. Interestingly the rat and human enzymes lack the carboxyl-terminal proline (residue 841) present in the rabbit enzyme and terminate at isoleucine (residue 840). The genetic basis for this difference in carboxyl termini is unknown. However, unlike the other amino acid changes, it cannot be accounted for by a single base-pair substitution. A comparison of the 3' untranslated regions in these cDNAs shows that there has been little constraint on the evolutionary divergence of most of this region (70% homology among the three species). There are, however, two repeated segments of DNA flanking the stop codons that are identical among all three species. Similar sequences are found within regions of DNA that contain a variety of transcriptional enhancers, suggesting the possibility that the repeats may be functional. SO - Eur J Biochem 1985 Oct 15;152(2):267-74. Neg-3. PMID:2372245 | PIR:A32541 B32541 TI - Rapid purification and characterization of histatins (histidine-rich polypeptides) from human whole saliva. AB - Three different polypeptides capable of stimulating histamine release from mast cells were isolated from human whole saliva, using heparin-gel chromatography followed by reversed-phase HPLC. The amino acid sequences of these peptides were shown to be identical to those of histatins 1, 3 and 5. SO - Arch Oral Biol 1990;35(6):415-9. Neg-4. PMID:2338067 | PIR:A31334 A31758 TI - Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences. AB - A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385). Placid, NY 12946. SO - Electrophoresis 1990 Feb;11(2):133-40. Neg-5. PMID:1322589 | PIR:TVFVMT TI - Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties. AB - RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity. Universitaire, Orsay, France. SO - Virology 1992 Aug;189(2):556-67. Neg-6. PMID:2825349 | PIR:TVHUJN TI - Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. AB - Nuclear oncogene products have the potential to induce alterations in gene regulation leading to the genesis of cancer. The biochemical mechanisms by which nuclear oncoproteins act remain unknown. Recently, an oncogene, v-jun, was found to share homology with the DNA binding domain of a yeast transcription factor, GCN4. Furthermore, GCN4 and the phorbol ester-inducible enhancer binding protein, AP-1, recognize very similar DNA sequences. The human proto-oncogene c-jun has now been isolated, and the deduced amino acid sequence indicates more than 80 percent identity with v-jun. Expression of cloned c-jun in bacteria produced a protein with sequence-specific DNA binding properties identical to AP-1. Antibodies raised against two distinct peptides derived from v-jun reacted specifically with human AP-1. In addition, partial amino acid sequence of purified AP-1 revealed tryptic peptides in common with the c-jun protein. The structural and functional similarities between the c-jun product and the enhancer binding protein suggest that AP-1 may be encoded by c-jun. These findings demonstrate that the proto-oncogene product of c-jun interacts directly with specific target DNA sequences to regulate gene expression, and therefore it may now be possible to identify genes under the control of c-jun that affect cell growth and neoplasia. California, Berkeley, CA 94720. SO - Science 1987 Dec 4;238(4832):1386-92. Neg-7. PMID:8179334 | PIR:A31780 TI - Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase mRNA and gene structure. AB - Previously we described the coding region of bovine heart Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase as determined from cDNA. We have isolated several overlapping clones and determined the DNAs which encode the entire mRNA of bovine heart bifunctional enzyme, including the 5'- and 3'-noncoding regions. the exon-intron structure of the 27-kb gene was determined and shown to consist of 16 exons. There are one primary and two secondary transcription initiation sites with the coding region beginning at exon 2. Exons 3-7 encode the Fructose 6-P,2-kinase domain, and exons 9-14 encode the Fructose 2,6-biphosphatase domain. Exon 15 is a unique exon for the heart type isozyme and contains the phosphorylation sites for protein kinases A and C. Alternative splicing of this exon accounts for the expression of two heart type isozymes in cardiac muscle. Exon 16 (also unique), 1820 bp long and located 7 kb downstream of exon 15, encodes the short C-terminus of the enzyme and is completely different from that of the rat heart bifunctional enzyme gene. Texas. SO - Arch Biochem Biophys 1994 May 1;310(2):467-74. Neg-8. PMID:3278933 | PIR:KBBOA2 TI - A new strategy for primary structure determination of proteins: application to bovine beta-casein. AB - A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C-terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS-PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N-terminal sequence of peptide (i-1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine beta-casein (209 residues) which was completely resequenced with only 239 Edman cycles. SO - FEBS Lett 1988 Mar 14;229(2):265-72. Neg-9. PMID:1939274 | PIR:ACRYD1 TI - Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide. AB - The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh. Columbia University, New York, New York 10032. SO - J Biol Chem 1991 Nov 25;266(33):22603-12. Neg-10. PMID:1668653 | PIR:KIZMPO TI - Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. AB - I describe here the organization of maize C4 chloroplast and non-C4 cytosolic pyruvate, orthophosphate dikinase (PPDK) genes and the molecular mechanisms underlying their differential expression. The maize C4 chloroplast PPDK gene (C4ppdkZm1) appears to have been created by the addition of an exon encoding the chloroplast transit peptide at a site upstream of a cytosolic PPDK gene (cyppdkZm1). A splice acceptor sequence located in the first exon of cyppdkZm1 allows the fusion of the transit peptide to the cyppdkZm1 sequences. A second cyPPDK gene (cyppdkZm2) shares extensive homology with cyppdkZm1 in the coding region and in the 5' flanking region up to the TATA box. By a novel protoplast transient expression method, I show that the light-inducible expression of C4ppdkZm1 is controlled by two expression programs mediated through separate upstream regulatory elements that are active in leaf, but inactive in root and stem. Light-mediated C4ppdkZm1 expression in maize is apparently uncoupled from leaf development and partially associated with chloroplast development. For cyppdkZm1 expression, distinct upstream elements and a specific TATA promoter element, located in the first intron of C4ppdkZm1, are required. The low expression of cyppdkZm2 can be attributed to an absence of upstream positive elements and weak activity of the TATA promoter element. SO - Plant Cell 1991 Mar;3(3):225-45. Neg-11. PMID:6754709 | PIR:B46111 CCBO JA0136 JQ1253 JQ1254 JS0090 OACH VCVXUV VCVXWL VCVXY1 TI - Micro-identification of amino-terminal acetylamino acids in proteins. AB - By using a reversed phase HPLC system, we have developed a rapid and quantitative micro-identification method for amino-terminal acetylamino acids in proteins at the sample range of 10--100 nmol. The process of the identification method consists of the following steps: (1) digestion of a protein by an appropriate protease; (2) separation of an acidic peptide(s) from the digest on a Dowex 50 x 2 column; (3) further purification of the acidic peptide(s) by reversed phase HPLC using a C18 column; (4) subsequent exhaustive digestion of each acidic peptide to amino acids and an acylamino acid, by pronase and carboxypeptidase Y; (5) isolation of the acylamino acid on a Dowex 50 x 2 column; (6) identification of the acylamino acid on a C18 column; (7) confirmation of the acyl group by HPLC as an 1-acyl-2-dansylhydrazine derivative and of the amino acid on amino acid analyzer. By applying this new method to such proteins as ovalbumin and cytochrome c, their amino-terminal acetylamino acids can be determined in the range of less than 10 nmol. SO - J Biochem (Tokyo) 1982 Sep;92(3):607-13. Neg-12. PMID:3755379 | PIR:KIRTC1 KIRTC2 TI - Cloning and expression of multiple protein kinase C cDNAs. AB - Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed. SO - Cell 1986 Aug 15;46(4):491-502. Neg-13. PMID:6952189 | PIR:EQBOA TI - Partial characterization of the mRNA that codes for enkephalins in bovine adrenal medulla and human pheochromocytoma. AB - [Met]enkephalin and [Leu]enkephalin are derived from a protein in bovine adrenal medulla that contains multiple copies of [Met]enkephalin [Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 3265--3268.] Here we characterize pro-enkephalin mRNA from bovine and human tissue by use of an oligodeoxynucleotide pentadecamer probe complementary to codons for Tyr-Gly-Gly-Phe-Met ([Met]enkephalin). This probe hybridizes specifically to a species of poly(A)-RNA from adrenal medulla and human pheochromocytoma, (1400--1450 bases), and also to [Met]enkephalin-containing pro-opiomelanocortin mRNAs from bovine pituitary (1200 bases) and from mouse pituitary tumor cell (1100 bases). A cloned cDNA probe (144 bases) complementary to the region of pro-opiomelanocortin mRNA that codes for lipotropin does not hybridize to the RNA from bovine adrenal medulla, demonstrating that the latter RNA is not pro-opiomelanocortin mRNA. The pentadecamer probe was extended to make cDNA with reverse transcriptase after hybridizing it to adrenal poly(A)-RNA. The sequence of an extended cDNA, 62 bases in length, was found to correspond exactly to that expected from the amino acid sequence of peptide E (a bovine adrenal peptide containing [Met]- and [Leu]enkephalin sequences). This cDNA also forms a specific hybrid with the RNA from bovine adrenal and human pheochromocytoma, confirming that these species of RNA are pro-enkephalin mRNA. SO - Proc Natl Acad Sci U S A 1982 Jan;79(2):360-4. Neg-14. PMID:6688355 | PIR:FGHUA TI - Characterization of a complementary deoxyribonucleic acid coding for the alpha chain of human fibrinogen. AB - A human liver cDNA library was screened for the alpha chain of fibrinogen with a cDNA clone from the corresponding bovine molecule as a hybridization probe. Several human clones coding for the alpha chain were identified, and one of these was used to rescreen the entire cDNA library of 18 000 recombinants. Plasmids with the largest cDNAs were isolated, and their inserts were sequenced. The largest cDNA insert contained 2224 base pairs, including a noncoding region at the 5'-end followed by a region coding for a signal peptide of 19 (or 16) amino acids and a mature protein of 625 amino acids, a stop codon of TAG, another noncoding region, and a poly(A) tail at the 3'-end. Eight tandem repeats of 39 base pairs were observed starting with nucleotide 905 (amino acid residue 270) and ending with nucleotide 1213 (amino acid residue 372). The identity in the nucleotide sequence in the tandem repeats ranged from 72 to 95% when compared to a consensus sequence. The predicted amino acid sequence for the mature polypeptide chain was 15 amino acids longer at the carboxyl-terminal end than that of the alpha chain isolated and sequenced from plasma fibrinogen. This indicates that minor proteolysis has taken place on the carboxyl-terminal end of the alpha chains, and this modification has probably occurred during secretion or circulation of the protein in plasma. SO - Biochemistry 1983 Jun 21;22(13):3237-44. Neg-15. PMID:6687628 | PIR:ISRBT MORBLD TMRBA TMRBB TPRBCS TPRBIS TPRBTS TI - A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing. AB - Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype. SO - Nature 1983 Apr 21;302(5910):718-21. Neg-16. PMID:2002045 | PIR:TIHUGK TI - Structure of the human lipoprotein-associated coagulation inhibitor gene. Intro/exon gene organization and localization of the gene to chromosome 2. AB - Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling. Medical Center, St. Louis, Missouri 63110. SO - J Biol Chem 1991 Mar 15;266(8):5036-41. Neg-17. PMID:3223413 | PIR:MMHUE4 TI - Expression of specific isoforms of protein 4.1 in erythroid and non-erythroid tissues. AB - Protein 4.1 in red cells is an important submembrane linking protein that binds to spectrin actin complexes at one end of its structure and to transmembrane proteins, such as glycophorin, at the other. Protein 4.1 thus contributes to the strength and flexibility of the erythrocyte membrane, a fact dramatically exemplified by the appearance of hereditary hemolytic anemias in patients with absent or abnormal protein 4.1. Recently, protein 4.1 forms have been discovered in many non-erythroid tissues. Their intracellular locations raise the possibility that these isoforms might have different functions. We have thus conducted comparative analysis of erythroid and non-erythroid protein 4.1 forms by cloning and sequencing erythroid and lymphoid protein 4.1 cDNAs. The lymphoid protein 4.1 isoforms exhibit at least five nucleotide sequence motifs that appear to be either inserted or deleted relative to the erythroid mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of this alternatively spliced mRNA during erythroid maturation implies the existence of a lineage specific splicing mechanism whose activity is triggered by terminal maturation. Two motifs alter the 5' untranslated region of the "prototypical" erythroid mRNA in such a way as to permit synthesis of a novel larger isoform. This form appears to localize preferentially in the nucleus. We thus conclude that a single gene gives rise to multiple protein 4.1 isoforms with potentially diverse locations and functions. Haven, CT 06510. SO - Adv Exp Med Biol 1988;241:81-95. Neg-18. PMID:1709100 | PIR:CGHU1V SGHU1V TI - Insulin binds to type V collagen with retention of mitogenic activity. AB - The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity. SO - Exp Cell Res 1991 Jun;194(2):180-5. Neg-19. PMID:7522053 | PIR:SGHU1V TI - Identification of a PAI-1 binding site in vitronectin. AB - Active PAI-1 (plasminogen activator inhibitor 1) is bound to vitronectin in plasma and in the extracellular matrix. In this study we aimed at identifying the PAI-1 binding site in vitronectin, which at present is a matter of dispute. Vitronectin was cleaved with trypsin and the fragments were tested for inhibitory effect on the PAI-1/vitronectin interaction using vitronectin-coated microtiter plates. Intact vitronectin and the tryptic digest of vitronectin both caused a 50% reduction in PAI-1 binding at a concentration of about 2 nmol/I. Gel-filtration on Sephadex G-50 superfine of the tryptic peptides resulted in one main peak of inhibitory activity. The elution volume, Kav, was 0.55 indicating (a) medium-size peptide(s). The peptide was further purified by reverse-phase HPLC. Structural analysis revealed that it constituted the 45 NH2-terminal amino-acid residues in vitronectin. The NH2-terminal vitronectin peptide caused a 50% decrease in PAI-1 binding to the vitronectin-coated microtiter plates at a concentration of about 13 nmol/l. Thus, the peptide is a little less effective in this respect than intact vitronectin. Reduced and S-carboxymethylated peptide had no effect on the interaction. The NH2-terminal vitronectin fragment increased the stability of active PAI-1 by about 60%, which is a little less than with intact vitronectin. The peptide also prevented PAI-1 from oxidation with chloramine T. The half-life was prolonged about 4-fold as compared to about 30-fold with intact vitronectin. SO - Biochim Biophys Acta 1994 Sep 21;1208(1):104-10. Neg-20. PMID:1712752 | PIR:A39901 TI - Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene. AB - The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse". Medical School, Imperial College, London, United Kingdom. SO - Genomics 1991 Jun;10(2):301-7. Neg-21. PMID:2997120 | PIR:MMECZB TI - Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins. AB - The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products. SO - J Bacteriol 1985 Nov;164(2):578-84. Neg-22. PMID:8294447 | PIR:F70584 TI - The immunodominant 38-kDa lipoprotein antigen of Mycobacterium tuberculosis is a phosphate-binding protein. AB - Several antigens of Mycobacterium tuberculosis have been identified by monoclonal antibodies and are being exploited in the development of improved vaccines and diagnostic reagents, but none has been linked to a specific function. Herein we report that the 38-kDa extracellular lipoprotein antigen, the most potent immunogen of the mycobacteria, is a phosphate-binding protein with features very similar to those of the well characterized periplasmic phosphate-binding protein of Escherichia coli which serves as an initial receptor for active transport. This is also the first report definitively linking a function of a binding protein anchored to a membrane and found in other than Gram-negative bacteria. Texas 77030. SO - J Biol Chem 1994 Jan 21;269(3):1956-8. Neg-23. PMID:2479556 | PIR:SGHU1V TI - Sulfation of two tyrosine-residues in human complement S-protein (vitronectin). AB - Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation. SO - Eur J Biochem 1989 Nov 6;185(2):391-5. Neg-24. PMID:2387866 | PIR:C1HURB C1HUS TI - Ca2+ binding properties and Ca2(+)-dependent interactions of the isolated NH2-terminal alpha fragments of human complement proteases C1-r and C1-s. AB - The NH2-terminal alpha fragments of human complement proteases C1-r and C1-s were obtained by limited proteolysis of the native proteins with trypsin, and isolated. C1-r alpha extended from residues 1 to 208 of C1-r A chain, with at least two cleavage sites within disulfide loops, after lysine 134 and arginine 202. C1-s alpha comprised residues 1-192 of the C1-s A chain, with one cleavage site within a disulfide loop, after arginine 186. C1-r alpha was monomeric either in the presence or absence of Ca2+ but formed Ca2(+)-dependent dimers with native C1-s. C1-s alpha dimerized in the presence of Ca2+ and formed Ca2(+)-dependent tetramers (C1-s alpha-C1-r-C1-r-C1-s alpha) with native C1-r. C1-r alpha and C1-s alpha associated in the presence of Ca2+ to form C1-r alpha-C1-s alpha heterodimers. Equilibrium dialysis studies indicated that each alpha region binds Ca2+ with a dissociation constant ranging from 19 microM (native proteins) to 38 microM (fragments). C1-r alpha, C1-r alpha-C1-s alpha, and the native C1-s-C1-r-C1-r-C1-s tetramer bound 0.9, 1.9, and 4.0 Ca2+ atoms/mol, respectively, whereas dimers C1-s alpha-C1-s alpha and C1-s-C1-s incorporated 2.9 and 3.0 Ca2+ atoms/mol. It is concluded that each alpha region contains one high affinity Ca2+ binding site. This 1:1 stoichiometry is maintained upon heterologous (C1-r-C1-s) interaction, whereas the homologous (C1-s-C1-s) interaction provides one additional binding site. Grenoble, France. SO - J Biol Chem 1990 Aug 25;265(24):14469-75. Neg-25. PMID:751625 | PIR:OACH TI - A correction and extension of the acetylated amino terminal sequence of ovalbumin. AB - The acetylpeptides derived from S-carboxymethylovalbumin by cyanogen bromide and chymotrypsin have been isolated and shown by enzyme digestion and the dansyl-Edman method to fit the sequence acetyl-Gly-Ser-Ile-Gly-Ala-Ala-Ser-Met-Glu-Phe. This corrects the order of the third and fourth residues in the five-residue sequence given by Narita and Ishii [J. Biochem. (Tokyo), 1962, 52, 367--73]. The overlap of the C-terminal sequence of this extended sequence with the six-residue N-terminal sequence surrounding a half-cystine residue in ovalbumin gives the N-terminal sequence for ovalbumin as acetyl-Gly-Ser-Ile-Gly-Ala-Ala-Ser-Met-Glu-Phe-Cys-Phe-Asp-Val-Phe-Lys with residue 11 a cysteine residue. SO - Aust J Biol Sci 1978 Oct;31(5):443-6. Neg-26. PMID:6628686 | PIR:S05207 TI - Amino acid sequence characterization of mammalian vimentin, the mesenchymal intermediate filament protein. AB - The amino-terminal 98 residues of porcine vimentin have been determined by amino acid sequence studies. Extensive overlap is seen with the corresponding region of the carboxyterminal 448 residues of hamster vimentin predicted from DNA sequence studies, which left the very amino-terminal region unknown. The combined data show that contrary to gel electrophoretic results, mammalian vimentin contains only about 467 residues, and that species-specific drift occurs mainly in the amino-terminal non-alpha-helical array. The results are discussed parallel to emerging concepts on intermediate filament protein diversity. SO - FEBS Lett 1983 Oct 31;163(1):22-4. Neg-27. PMID:6575389 | PIR:FGHUA FGHUG TI - Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications. AB - Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene. SO - Proc Natl Acad Sci U S A 1983 Jul;80(13):3953-7. Neg-28. PMID:1699599 | PIR:A35702 TI - Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology. AB - Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes. SO - Biochemistry 1990 Aug 14;29(32):7420-5. Neg-29. PMID:3036070 | PIR:C1HURB TI - Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r. AB - The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r. SO - Biochem J 1987 Feb 1;241(3):711-20. Neg-30. PMID:1716243 | PIR:A40303 TI - Molecular cloning and sequence analysis of the murine cDNA for the cystic fibrosis transmembrane conductance regulator. AB - We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common delta F508 mutation were conserved between human and mouse. Texas 77030. SO - Genomics 1991 Jul;10(3):547-50. Neg-31. PMID:1167550 | PIR:HSBO3 TI - Histone III. VI. Two forms of calf thymus histone III. AB - Microheterogeneity in the amino acid sequence of pea embryo histone III at residue 96 has been established previously. It has been indicated that calf-thymus contains two forms of histone III, with 1 or 2 residues of cysteine, respectively. Evidence is presented here that these two forms are also due to microheterogeneity at residue 96 with one form containing a cysteine residue and the other a serine residue. SO - J Biol Chem 1975 Mar 10;250(5):1919-20. Neg-32. PMID:2303595 | PIR:A32541 B32541 TI - Structural relationship between human salivary histatins. AB - Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group of polypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies. Massachusetts. SO - J Dent Res 1990 Jan;69(1):2-6. Neg-33. PMID:2859121 | PIR:INHUR TI - The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. AB - A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19. SO - Cell 1985 Apr;40(4):747-58. Neg-34. PMID:2226790 | PIR:TPHUIC TI - Molecular cloning of human cardiac troponin I using polymerase chain reaction. AB - We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart. London, UK. SO - FEBS Lett 1990 Sep 17;270(1-2):57-61. Neg-35. PMID:1934363 | PIR:TPHUIC TI - Troponin I isoform expression in human heart. AB - Troponin I is the inhibitory component of troponin, the thin filament regulatory complex in striated muscle. Separate genes encode cardiac-specific fast and slow skeletal-specific isoforms of this protein. We have previously described gene switching from the slow skeletal to the cardiac troponin I mRNA expression in developing rat heart. The purpose of this work was to characterize the expression of the different troponin isoforms in the human heart. Human cardiac and slow skeletal troponin I cDNA probes were obtained by screening an adult cardiac cDNA library and by Taq polymerase amplification of RNA from an infant's heart, respectively. We found that the cardiac troponin I isoform is tissue-specific in its expression in normal adult tissues. RNA blot analysis of cardiac ventricular RNA from infants with congenital heart disease and from an adult with cardiomyopathy revealed expression of human cardiac troponin I in all analyzed specimens. In addition, we found expression of slow skeletal troponin I mRNA and protein in infant hearts but no detectable mRNA expression in the adult heart. We conclude that troponin I isoforms are developmentally regulated in the human heart by a mechanism similar to that in the rat heart. of Medicine, St. Louis, Mo. SO - Circ Res 1991 Nov;69(5):1409-14. Neg-36. PMID:1409665 | PIR:A31334 A31758 TI - Farnesylcysteine, a constituent of the alpha and beta subunits of rabbit skeletal muscle phosphorylase kinase: localization by conversion to S-ethylcysteine and by tandem mass spectrometry. AB - The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed. Federal Republic of Germany. SO - Proc Natl Acad Sci U S A 1992 Oct 15;89(20):9554-8. Neg-37. PMID:3477803 | PIR:MWAXIC TI - The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. AB - Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. We now present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which we deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approximately 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approximately 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an alpha-helical, coiled-coil structure. We conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, we find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. We discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure. Bethesda, MD 20892. SO - Proc Natl Acad Sci U S A 1987 Oct;84(19):6720-4. Neg-38. PMID:2153895 | PIR:A60049 TI - Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis. AB - Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes. 97201. SO - Brain Res Mol Brain Res 1990 Jan;7(1):61-8. Neg-39. PMID:2757638 | PIR:A33430 TI - Caldesmon has two calmodulin-binding domains. AB - Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. C. & Bryan, J., J. Biol. Chem. 261, 16155-16160, 1987; Yazawa, M., Yagi, K. & Sobue, K., J. Biochem. 102, 1065-1073, 1987). In the case of CNBr cleavage a 37 kDa calmodulin-binding fragment (CB40) was obtained. Both CT40 and CB40 contain a reactive thiol group, but these thiols are apparently in different environments as judged by the responses of attached fluorescent labels to calmodulin-binding. A comparison of the N-terminal sequences of CB40 and CT40 with the complete sequence of caldesmon shows that the two calmodulin-binding fragments in fact originate from different parts of the parent molecule. Thus there exist two calmodulin-binding sites in caldesmon, one in the N-terminal half and the other in the C-terminal half of the molecule. This is consistent with the recent finding that up to two calmodulin molecules can be crosslinked to each caldesmon molecule (Wang, C.-L.A., Biochem. Biophys. Res. Commun., 156, 1033-1038, 1988). 02114. (4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole) SO - Biochem Biophys Res Commun 1989 Jul 31;162(2):746-52. Neg-40. PMID:8636990 | PIR: TI - Deletion analysis of the che operon in the archaeon Halobacterium salinarium. AB - Halobacterium salinarium is a chemo- and phototactic archaeon whose signal transduction pathway includes the classical two-component system made up of CheA and CheY. Deletion analysis of the che operon in H. salinarium has been undertaken. Following the removal of the entire operon, the importance of each of the four individual members, cheY, cheB, cheA, and the novel member cheJ, was evaluated by their replacement in combinations of three. The mutant strains were investigated for their motility, their chemo- and phototactic signalling, and the rotational bias of their flagella. Loss of cheA, cheY or cheB led to the complete loss of chemo- and phototaxis, whereas the absence of cheJ caused a reduction in chemo- and phototactic ability. Reverse swimming and counterclockwise rotation of the flagella required the presence of cheA and CheY. The wild-type 50:50 distribution of forward and reverse swimming was observed in the strain lacking cheB, whereas this distribution was perturbed to 88:12 in the strain lacking cheJ. These results are compared with the corresponding deletion strains in Escherichia coli and provide new insights into the eu- and archeabacterial flagellar switch. SO - J Mol Biol 1996 May 17;258(4):548-54. Neg-41. PMID:1899185 | PIR:KABOSB TI - Specificity of two genetically related cell-envelope proteinases of Lactococcus lactis subsp. cremoris towards alpha s1-casein-(1-23)-fragment. AB - The specificity of two genetically related cell-envelope serine proteinases (PI-type and PIII-type) of Lactococcus lactis subsp. cremoris towards the alpha s1-casein-(1-23)-fragment, an important intermediate product of primary chymosin-directed proteolysis in cheese, has been established. Both enzymes showed, at pH 6.5 and under relatively low-ionic-strength conditions, a characteristic, mutually different, cleavage pattern that seems, in the first instance, to be determined by the charge N-terminal to the cleaved bond. With Pi, three cleavage sites were found in the N-terminal positively charged part of the peptide and, with PIII, three sites were found in the C-terminal negatively charged part. Comparison of the specific cleavage sites in this peptide and those in beta-casein revealed similarities with respect to the different residues which can occur N-terminally to the cleaved bond. The properties of these substrate residues match with the structural and various interactive features of the respective binding regions of the enzymes predicted on the basis of a close sequence similarity of the lactococcal proteinases with the subtilisin family. A hydrophobic interaction and/or hydrogen-bridge formation seems to govern the binding of the first amino acid residue N-terminal to the scissile bond. The more distantly N-terminally positioned sequence of residues apparently is attracted electrostatically by a negative charge in the binding region of PI and by a positive charge in that of PIII, provided that the opposite charge is is present at the appropriate position in this sequence. Hence a specific electrostatic binding may occur; additionally, hydrophobic interaction and/or hydrogen-bond formation is important. SO - Biochem J 1991 Jan 1;273(Pt 1):135-9. Neg-42. PMID:1935977 | PIR:PEMQAJ PEMQCJ S19682 S19684 TI - Development-dependent expression of isozymogens of monkey pepsinogens and structural differences between them. AB - The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development. Japan. SO - Eur J Biochem 1991 Nov 15;202(1):205-15. Neg-43. PMID:2369896 | PIR:INHUR TI - Functionally distinct insulin receptors generated by tissue-specific alternative splicing. AB - Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues. CA 90480. SO - EMBO J 1990 Aug;9(8):2409-13. Neg-44. PMID:692731 | PIR:OACH TI - Nucleotide sequence homology at 12 intron--exon junctions in the chick ovalbumin gene. AB - A short partial sequence homology is present at all intron-exon junctions, or splice points, in the chick ovalbumin gene; it is probably a signal for a splicing enzyme. The significance of the junction sequences for splicing is discussed. We find no evidence of strong Watson-Crick base pairing between adjacent junctions. SO - Nature 1978 Oct 12;275(5680):510-3. Neg-45. PMID:146828 | PIR:TPRBIS TI - Comparison of amino acid sequence of troponin I from different striated muscles. AB - The sequence of troponin I from fast and slow skeletal and cardiac muscle shows strong homology in the region which binds to actin and is responsible for inhibition of the actomyosin AT Pase. More differences are found in the N-terminal region which binds to troponin C. SO - Nature 1978 Jan 5;271(5640):31-5. Neg-46. PMID:518845 | PIR:FGHUA TI - Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites. AB - Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain. SO - Biochemistry 1979 Nov 27;18(24):5405-10. Neg-47. PMID:518846 | PIR:FGHUA TI - Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence. AB - The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone. SO - Biochemistry 1979 Nov 27;18(24):5410-6. Neg-48. PMID:8406024 | PIR:TPHUIC TI - Cloning and expression in Escherichia coli of the cDNA encoding human cardiac troponin I. AB - We have cloned and sequenced the human cardiac troponin I (cTnI)-encoding cDNA with the aim of expressing the cDNA in Escherichia coli. The cDNA was successfully expressed as a fusion product with beta-galactosidase and as an unfused protein. Both polypeptides were recognised by an anti-human cTnI antibody. Marischal College, Scotland. SO - Gene 1993 Sep 15;131(2):287-92. Neg-49. PMID:2701943 | PIR:A32794 TI - Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y. AB - The high-mobility-group protein HMG-I is a well-characterized nonhistone chromosomal protein that is preferentially expressed in rapidly dividing cells, binds to A. T-rich regions of DNA in vitro, and has been localized to particular regions of mammalian metaphase chromosomes. We isolated eight cDNA clones encoding HMG-I and its isoform HMG-Y from a human Raji cell cDNA library and detected blocks of nucleotide sequence rearrangements in the 5'-untranslated regions of these clones. In addition to this leader sequence variation, five of the eight cDNA clones had either a 33- or 36-base-pair in-frame deletion in their open reading frame (ORF); we found that this shortened ORF encodes the HMG-Y protein isoform. We present evidence that the 5'-untranslated-region and ORF heterogeneity of the cDNA clones is the result of alternative processing of RNA transcripts from a single functional gene. Several additional but probably nonfunctional HMG-I or HMG-Y gene copies exist in the human genome; we isolated and partially sequenced one of these pseudogenes and found that it is a processed HMG-Y retropseudogene. 99164. SO - Mol Cell Biol 1989 May;9(5):2114-23. Neg-50. PMID:2983222 | PIR:INHUR TI - Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. AB - We have deduced the entire 1,370-amino-acid sequence of the human insulin receptor precursor from a single complementary DNA clone. The precursor starts with a 27-amino-acid signal sequence, followed by the receptor alpha-subunit, a precursor processing enzyme cleavage site, then the beta-subunit containing a single 23-amino-acid transmembrane sequence. There are sequence homologies to human epidermal growth factor receptor and the members of the src family of oncogene products. SO - Nature 1985 Feb 28-Mar 6;313(6005):756-61. Neg-51. PMID:3208764 | PIR:KKBOB TI - Isolation and characterization of the bovine kappa-casein gene. AB - 1. The bovine kappa-casein gene has been isolated as a series of overlapping lambda clones and shown to consist of five exons distributed over a total length of approximately 13 kb. Most of the mature protein-coding sequence is contained in a single large exon. 2. Approximately 65% of the gene has been sequenced together with portions of the 5'- and 3'-flanking sequences. The immediate 5'-flanking sequence contains several motifs which are characteristic of upstream regions including a TATA box, a CAAT box, a sequence similar to that recognized by transcription factor AP-1 and a purine-rich sequence resembling that found upstream in all other lactoprotein genes. Other possible regulatory sequences are found upstream of exon 4. 3. The organization of the kappa-casein gene, together with its upstream sequence, confirms previous conclusions that it is unrelated to the calcium-sensitive-casein gene family to which it is linked. Evidence is presented which supports a previous suggestion that kappa-casein and the fibrinogens are evolutionarily related. 4. Intron sequences contain several examples of the A family of the artiodactyl Alu-like repeated sequences, together with a single example of a C-family sequence. The remainders of the introns of the kappa-casein gene, compared with the repeat elements and exons, are A + T-rich. 5. Among the lambda clones isolated, representatives were found of the A and B genetic variants which can be distinguished by restriction-enzyme analysis. Several other examples of polymorphisms in the non-coding region were found. SO - Eur J Biochem 1988 Dec 15;178(2):395-401. Neg-52. PMID:1339446 | PIR:TPRBIS TI - Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. AB - A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities. School of Medicine, Florida 33101. SO - J Biol Chem 1992 Dec 15;267(35):25407-13. Neg-53. PMID:3680248 | PIR:INHUR TI - Characterization of the promoter region of the human insulin receptor gene. Evidence for promoter activity. AB - Recombinant clones containing the promoter region of the human insulin receptor gene were isolated from genomic libraries derived from nondiabetic persons. A 1.5-kilobase pair fragment of the 5'-flanking region was sequenced. One transcriptional start site, located at 203 bases upstream from the start of translation was identified by nuclease S1 mapping and the primer extension experiment using the human insulin receptor mRNA. The bacterial chloramphenicol acetyltransferase assay revealed that a 573-base pair fragment immediately preceding the ATG has promoter activity and that the transcript initiates from the normal start site of the insulin receptor gene in the COS cells. The promoter region contains neither a "TATA box" nor a "CAAT box," has an extremely high G + C content, and contains seven central components of potential Sp 1 binding sites (GGGCGG or CCGCCC). These features are common to those found in the regulatory regions of a class of constitutively expressed "housekeeping" genes. A comparison between the promoter sequence of the human insulin receptor and those of other "housekeeping" genes revealed the presence of homologous sequences among these genes, in addition to the potential Sp 1 binding sites. SO - J Biol Chem 1987 Nov 25;262(33):16186-91. Neg-54. PMID:8612645 | PIR:SGHU1V TI - Limited plasmin proteolysis of vitronectin. Characterization of the adhesion protein as morpho-regulatory and angiostatin-binding factor. AB - The adhesion protein vitronectin is associated with extracellular matrices and serves as cofactor for plasminogen-activator inhibitor-1. Limited proteolysis by plasmin converts vitronectin into defined fragments which are detectable at sites of inflammation and angiogenesis. The loss and gain of binding functions of vitronectin fragments for macromolecular ligands was characterized in the present study. The initially generated 61--63-kDa vitronectin-(1--348)-fragment serves as typical binding component for plasminogen and binding function was lost upon carboxypeptidase B treatment indicating the importance of a C-terminal lysine. Complementary binding sites reside in isolated plasminogen kringles 1--3 (designated angiostatin) as deduced from direct binding and ligand blotting experiments. A synthetic vitronectin-(331--348)-peptide from the C-terminus of the 61--63-kDa fragment could mimic plasminogen and angiostatin binding. Also, the immobilized peptide bound tissue plasminogen-activator and mediated plasmin formation, comparable to fibrinogen-derived peptides. The 61--63-kDa vitronectin fragment was indistinguishable in its adhesive properties to intact vitronectin and bound active but not latent plasminogen-activator inhibitor-1. Late plasminolysis of vitronectin resulted in the processing of the N-terminal region of the protein with the generation of 42 kDa/35-kDa fragments that had Gly89 as new N-terminus and that were ineffective in promoting cell adhesion. Thus, at sites of cell-matrix interactions which become proteolytically modified by plasmin during inflammatory and angiogenic processes, vitronectin serves as plasminogen/angiostatin-binding factor. Due to this differential change in functions particularly at sites of deposition in the vascular system or at wound sites vitronectin is considered to be an important morpho-regulatory factor. SO - Eur J Biochem 1996 Mar 1;236(2):682-8. Neg-55. PMID:3780752 | PIR:FNBO TI - Complete primary structure of bovine plasma fibronectin. AB - The primary structure of the 2265 residues of bovine plasma fibronectin has been completed. The new sequences reported in this paper are residues 600-868 (269 residues), 1138-1217 (80 residues), 1518-1599 (82 residues) and 1868-2061 (194 residues). These sequences constitute six type III homology units and two non-homologous connecting strands. Thus, there are fifteen type III homology units in plasma fibronectin. Evidence for two of the three splice variants found in rat liver cells [Schwarzbauer et al. (1983) Cell 35, 421-431] was obtained. No indication of the 'extra' type III domain present in some human fibroblast fibronectins [Kornblihtt et al. (1984) EMBO J. 3, 221-226] was found. Three carbohydrate groups (two glucosamine-based and one galactosamine-based) were identified, giving a total of eight carbohydrate groups in the longest splice variant of bovine plasma fibronectin. A second free sulfhydryl group (cysteine) was identified. This cysteine, like the first, is in a type III homology unit. HPLC patterns of peptides obtained from the C-terminal 6-kDa fragment suggested that the interchain bridge pattern of fibronectin is antiparallel. The bovine plasma fibronectin sequence is highly homologous to the human cellular fibronectin sequence deduced from the cDNA sequence [Kornblihtt et al. (1985) EMBO J. 4, 1755-1759]. SO - Eur J Biochem 1986 Dec 1;161(2):441-53. Neg-56. PMID:7543369 | PIR:A56968 S34127 TI - X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. AB - The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506. USA. SO - Cell 1995 Aug 11;82(3):507-22. Neg-57. PMID:632262 | PIR:FGHUA TI - Localization of the alpha-chain cross-link acceptor sites of human fibrin. AB - The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain. SO - J Biol Chem 1978 Apr 10;253(7):2184-95. Neg-58. PMID:1597484 | PIR:OACH TI - Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link. AB - The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin. SO - J Biol Chem 1992 Jun 5;267(16):11565-72. Neg-59. PMID:2882469 | PIR:WHRTY TI - Identification and cell type specificity of the tyrosine hydroxylase gene promoter. AB - Genomic DNA encoding the rat tyrosine hydroxylase (TH) gene was isolated from a lambda phage library using a nick-translated fragment from a cDNA clone for rat TH. We have determined the initiation site for TH RNA synthesis and have sequenced 1100 bases of the primary transcript and 5' flanking region. The 5' end of the transcript is the same in several rat tissues in which TH is expressed as well as in rat pheochromocytoma cells (PC). RNA prepared from PC cells that had been stimulated with dexamethasone also mapped to the same transcription start site. Sequence upstream from the initiation site contains the canonical TATA box, but no apparent CAAT box. When a portion of the 5' flanking region of the TH gene (-773 to + 27) is fused to the chloramphenicol acetyltransferase (CAT) gene, it promotes expression of CAT in pheochromocytoma cells and GH4 cells, but not in two neural tumour lines, RT4-D and B103, nor in several non neural cell lines. This suggests that this region of the TH gene has features that confer tissue-restricted expression on the TH promoter. SO - Nucleic Acids Res 1987 Mar 11;15(5):2363-84. Neg-60. PMID:2876781 | PIR:DVHU1 TI - Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. AB - Resistance of tumor cells to multiple cytotoxic drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human cells is determined by the mdr1 gene, encoding a high molecular weight membrane glycoprotein (P-glycoprotein). Complete primary structure of human P-glycoprotein has been determined from the cDNA sequence. The protein, 1280 amino acids long, consists of two homologous parts of approximately equal length. Each half of the protein includes a hydrophobic region with six predicted transmembrane segments and a hydrophilic region. The hydrophilic regions share homology with peripheral membrane components of bacterial active transport systems and include potential nucleotide-binding sites. These results are consistent with a function for P-glycoprotein as an energy-dependent efflux pump responsible for decreased drug accumulation in multidrug-resistant cells. SO - Cell 1986 Nov 7;47(3):381-9. Neg-61. PMID:3510184 | PIR:QRECCS QRECCY QRECCZ TI - Nucleotide sequence corresponding to five chemotaxis genes in Escherichia coli. AB - The nucleotide sequence of DNA which contains five chemotaxis-related genes of Escherichia coli, cheW, cheR, cheB, cheY, and cheZ, and part of the cheA gene was determined. Molecular weights of the polypeptides encoded by these genes were calculated from translated amino acid sequences, and they were 18,100 for cheW, 32,700 for cheR, 37,500 for cheB, 14,100 for cheY, and 24,000 for cheZ. Nucleotide sequences which could act as ribosome-binding sites were found in the upstream region of each gene. After the termination codon of the cheW gene, a typical rho-independent transcription termination signal was observed. There are no other open reading frames long enough to encode polypeptides in this region except those which code for the two previously reported genes tar and tap. SO - J Bacteriol 1986 Jan;165(1):161-6. Neg-62. PMID:1372900 | PIR:S06102 TI - Mutations away from splice site recognition sequences might cis-modulate alternative splicing of goat alpha s1-casein transcripts. Structural organization of the relevant gene. AB - alpha s1-Casein variants F and D, synthesized in goat milk at lower levels than variant A, essentially differ from it by internal deletions of 37 and 11 amino acid residues, respectively. Northern blot analysis of mRNAs encoding alpha s1-casein F and A and sequencing of the relevant cloned cDNAs, as well as sequencing of in vitro amplified genomic fragments, revealed multiple alternatively processed transcripts, from the F allele. Although correctly spliced messengers were identified, most of the FmRNAs lacked three exons. These exons, further identified as exons 9, 10, and 11, together encode the 37 amino acid residues present in alpha s1-casein variant A but missing in variant F. Exon 9 codes for the sequence present in variant A but deleted in variant D. A single nucleotide deletion in exon 9 and two insertions, 11 and 3 base pairs in length, in the downstream intron, were identified as mutations potentially responsible for the alternative skipping of these 3 exons. From a computer-predicted secondary structure it appeared that the 11-base pair insertion might be involved in base-pairing interactions with the intron 5' splice site which might consequently be less accessible to U1 snRNA. We also report here the complete structural organization of the goat alpha s1-casein transcription unit, deduced from polymerase chain reaction experiments. It contains 19 exons scattered within a nucleotide stretch nearly 17-kilobase pairs long. Agronomique, Jouy-en-Josas, France. SO - J Biol Chem 1992 Mar 25;267(9):6147-57. Neg-63. PMID:6321434 | PIR:BYECPR TI - Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene. AB - The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively. SO - J Bacteriol 1984 Mar;157(3):772-8. Neg-64. PMID:3015680 | PIR:PHRBG TI - Complete cDNA sequence for rabbit muscle glycogen phosphorylase. AB - The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed. SO - FEBS Lett 1986 Aug 18;204(2):283-7. Neg-65. PMID:2546820 | PIR:OKBOG TI - The cDNA of the two isoforms of bovine cGMP-dependent protein kinase. AB - cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb. Saarlandes, Homburg/Saar, FRG. SO - FEBS Lett 1989 Jul 17;251(1-2):191-6. Neg-66. PMID:3271384 | PIR:KBBOA2 TI - Complete nucleotide sequence of the bovine beta-casein gene. AB - The beta-casein gene is a member of a small gene family encoding the calcium-sensitive caseins, which are specifically synthesized and secreted by the mammary gland during lactation in response to both peptide and steroid hormones. The caseins are involved in the transport of calcium phosphate in milk, which is important for bone development in the infant mammal. We report here the organization and complete DNA sequence of the 8.5 kb long bovine beta-casein gene. Comparison with the rat beta-casein gene reveals that the exons of both genes correspond exactly. The 5' flanking sequences of all Ca-sensitive casein genes are conserved within the proximal 200 bp and contain several elements that probably function as cis-acting regulatory elements, including an octamer-like motif, and SV40-type core enhancer and a sequence that appears to be common to all lactoprotein genes. The latter sequence is flanked on either side by 12 bp direct repeats. These direct repeats are themselves each part of sequences that display two-fold symmetry. The first 30 nucleotides of the 3' flanking regions in the bovine and rat beta-caseins are well conserved, indicating that they are likely to be involved in the mechanism of 3' end processing of the primary transcript. SO - Aust J Biol Sci 1988;41(4):527-37. Neg-67. PMID:2414098 | PIR:SGHU1V TI - Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin. AB - cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition. SO - EMBO J 1985 Oct;4(10):2519-24. Neg-68. PMID:2808371 | PIR:MMHUE4 TI - O-N-acetyl-D-glucosamine moiety on discrete peptide of multiple protein 4.1 isoforms regulated by alternative pathways. AB - Erythrocyte protein 4.1 is a cytoplasmic protein that possesses a protein-saccharide modification structure, an O-N-acetyl-D-glucosamine (GlcNAc) moiety. We determined the amino acid sequence of the proteolytic fragment containing the O-GlcNAc moiety after labeling the saccharide with [3H]galactose in the presence of bovine milk galactosyltransferase. Glycosylation appears to occur on one or more serine or threonine residues in the following sequence: Thr-Ala-Gln-Thr-Ile-Thr-Ser-Glu-Thr-Pro-Ser-Ser-Thr-Thr-Thr-Thr-Gln-Ile-Th r-Lys . This sequence corresponds to the carboxyl-terminal half of the 34-amino acid peptide in the 22/24-kDa carboxyl-terminal domain of protein 4.1, which is one of the discrete peptides regulated by alternative RNA splicing. Multiple protein 4.1 isoforms in erythroid and nonerythroid cells including major components of erythrocyte membrane proteins, 4.1a and 4.1b, appear to contain this sequence since most immunochemically reactive proteins were labeled with [3H]galactose, with the exception of several variant polypeptides. These results appear to suggest the functional or biological significance of the O-GlcNAc linkage in protein 4.1. Medicine, Hokkaido University, Sapporo, Japan. SO - J Biol Chem 1989 Oct 25;264(30):18149-55. Neg-69. PMID:2215649 | PIR:BYECPR TI - High specificity of a phosphate transport protein determined by hydrogen bonds. AB - Transport of the essential nutrient phosphorus--primarily in the form of orthophosphate--into cells and organelles is highly specific. This is exemplified by the uptake of phosphate or its close analogue arsenate by bacterial cells by way of a high affinity active transport system dependent on a phosphate-binding protein; this system is unable to recognize other inorganic oxyanions and is, moreover, distinct from the one for sulphate transport. The phosphate-binding protein is a member of a family of periplasmic proteins acting as initial high-affinity receptors for the osmotic shock-sensitive active transport systems or permeases for various sugars, amino acids, oligopeptides, and oxyanions. We report here the highly refined 1.7 A resolution X-ray structure of the liganded form of the phosphate-binding protein. The structure reveals the atomic features responsible for phosphate selectivity, either in monobasic or dibasic form, and the exclusion of sulphate. These features are fundamental to understanding phosphate transport systems and molecular recognition of charged substrates or ions in other biological processes. Texas 77030. SO - Nature 1990 Sep 27;347(6291):402-6. Neg-70. PMID:2494172 | PIR:PEPG TI - Synthesis, purification, and active site mutagenesis of recombinant porcine pepsinogen. AB - In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active. Oklahoma City. SO - J Biol Chem 1989 Mar 15;264(8):4482-9. Neg-71. PMID:1993173 | PIR:TIHUGK TI - Intron-exon organization of the human gene coding for the lipoprotein-associated coagulation inhibitor: the factor Xa dependent inhibitor of the extrinsic pathway of coagulation. AB - Blood coagulation can be initiated when factor VII(a) binds to its cofactor tissue factor. This factor VIIa/tissue factor complex proteolytically activates factors IX and X, which eventually leads to the formation of a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits factor Xa directly and, in a Xa-dependent manner, also inhibits the factor VIIa/tissue factor complex. Here we report the cloning of the human LACI gene and the elucidation of its intron-exon organization. The LACI gene, which spans about 70 kb, consists of nine exons separated by eight introns. As has been found for other Kunitz-type protease inhibitors, the domain structure of human LACI is reflected in the intron-exon organization of the gene. The 5' terminus of the LACI mRNA has been determined by primer extension and S1 nuclease mapping. The putative promoter was examined and found to contain two consensus sequences for AP-1 binding and one for NF-1 binding, but no TATA consensus promoter element. Netherlands. SO - Biochemistry 1991 Feb 12;30(6):1571-7. Neg-72. PMID:2773933 | PIR:A32541 TI - Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs. AB - A cDNA coding for histatin 1 was isolated from a human submandibular-gland library and sequenced. This cDNA was used to probe RNAs isolated from a variety of tissues to investigate tissue-specific regulation and to determine whether histatins might play a role other than in the oral cavity. The same probe was also used for Southern blot analysis of human genomic DNA restricted with various enzymes, and it showed that the genes coding for histatins are on the same chromosome. In situ hybridization of the cDNA probe to metaphase chromosome spreads was performed to determine chromosomal location of the genes for histatins. A genomic fragment isolated using the cDNA probe was also hybridized to chromosome spreads, and the same chromosome was identified. The genes for histatins are located on chromosome 4, band q13. We have shown that three histatin mRNAs are expressed in human parotid and submandibular glands but in none of the other tissues studied. These results suggest that histatins are specific to salivary secretions. 02118. SO - Am J Hum Genet 1989 Sep;45(3):381-7. Neg-73. PMID:7925354 | PIR:UDCH TI - Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin. AB - Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin. Klinik und Poliklinik, LMU Munchen, Germany. SO - Eur J Biochem 1994 Sep 1;224(2):407-15. Neg-74. PMID:1967175 | PIR:DVHU1 TI - Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins. AB - The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication. SO - J Biol Chem 1990 Jan 5;265(1):506-14. Neg-75. PMID:1646596 | PIR:TPHUN1 TI - Purification and characterization of a higher-molecular-mass form of protein phosphotyrosine phosphatase (PTP 1B) from placental membranes. AB - Purification of a major placental membrane protein phosphotyrosine phosphatase (PTP-I) through the use of a nonhydrolysable phosphotyrosine analogue affinity ligand has enabled identification of the enzyme as a single polypeptide of at least 46 kDa. This phosphatase specifically dephosphorylates phosphotyrosine-containing substrates, including the src peptide, the epidermal-growth-factor receptor tyrosine kinase and the non-receptor tyrosine kinase p56lck. The p56lck can be dephosphorylated by PTP-I at two tyrosine residues (Tyr-394 and Tyr-505), which are differentially phosphorylated in vitro and in vivo and have been suggested to modulate kinase activity. The activity of PTP-I towards these substrates indicates a possible function of regulation of cellular tyrosine phosphorylation pathways at the level of growth-factor receptor and/or oncogene/proto-oncogene tyrosine kinases. Kinetic analyses show that PTP-I exhibits a Km value of about 2 microM with either src peptide or reduced, carboxyamidomethylated and maleylated (RCM)-lysozyme as substrate, and is inhibited in a mixed competitive manner by the polyanions heparin and poly(Glu4,Tyr1). Sequencing of PTP-I peptides reveals almost complete identity with sequences within the N-terminal half of the 37 kDa non-receptor tyrosine phosphatase 1B. However, the size and amino acid composition of PTP-I are similar to that of a higher-molecular-mass form of PTP 1B predicted from cDNA cloning. These results suggest that the 37 kDa PTP 1B is a proteolysed form of PTP-I, and provide evidence that a larger form of PTP 1B exists in vivo, at least in association with placental membranes. SO - Biochem J 1991 Jun 1;276 ( Pt 2):315-23. Neg-76. PMID:7819249 | PIR:I38344 TI - Dissecting titin into its structural motifs: identification of an alpha-helix motif near the titin N-terminus. AB - Titin, also known as connectin, is a giant modular protein specifically found in vertebrate striated muscle. Since the huge size of titin does not allow a direct structure determination, we have started a long-term project to characterize the protein by cutting it into smaller domains or structural units. The major part of the titin sequence is assembled by modules approximately 100 amino acids long that belong to two major protein superfamilies. Most of these modules are linked together by stretches of variable length with unique sequence. No direct structural characterization has been achieved so far for any of these linkers. We present here a study of a stretch located in the titin N-terminus and part of a linker between two modules. Our attention was drawn toward this region because it shows 100% probability to form a coiled coil when analyzed by a prediction program. A synthetic 38 amino acid peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultracentrifugation at various pH, salt, and peptide concentrations. Under all conditions, it shows a strong tendency to form alpha-helical structures. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate breaks, and the titin peptide is a monomeric helix in equilibrium with its random coil conformation. We discuss the factors which stabilize the helical conformation and the possible role of this stretch in vivo. SO - Biochemistry 1995 Jan 17;34(2):553-61. Neg-77. PMID:3403514 | PIR:HSHUP2 TI - Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines. AB - Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3. Institut de Recherches sur le Cancer, Lille, France. SO - J Biol Chem 1988 Aug 15;263(23):11059-62. Neg-78. PMID:2022644 | PIR:MMHUE4 TI - Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1. AB - Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis. University of California, Berkeley 94720. SO - J Biol Chem 1991 May 5;266(13):8273-80. Neg-79. PMID:3030286 | PIR:C1HURB TI - Cloning and sequencing of full-length cDNA encoding the precursor of human complement component C1r. AB - The sequencing of human liver cDNA clones encoding the entire C1r precursor protein has confirmed the previously determined peptide sequence and has shown that there is a leader peptide which is 17 amino acids long. A residue tentatively identified as beta-hydroxyaspartic acid [Arlaud, Willis & Gagnon (1986) Biochem. J., in the press] located in the C1r A-chain, within an epidermal-growth-factor consensus sequence, was found to be encoded as asparagine. Two sequence elements, tandemly located in the A-chain, are related to a sequence widespread among proteins which interact with C3b or C4b. Structural comparisons between different clones indicate that multiple polyadenylation sites are responsible for the length heterogeneity observed for C1r mRNA from liver and Hep G2 cells. SO - Biochem J 1986 Dec 15;240(3):783-7. Neg-80. PMID:2216462 | PIR:GEHUM TI - Overexpression of matrix Gla protein mRNA in malignant human breast cells: isolation by differential cDNA hybridization. AB - Genetic alterations are involved in the development of human breast cancer. We sought to isolate genes that are differentially expressed or suppressed in cultured human breast carcinoma cells as compared to cultured normal human breast epithelial cells by employing differential screening of selected cDNA libraries. Analysis of several clones thus isolated revealed that the matrix Gla protein (MGP) gene is overexpressed in the breast cancer cell line 600 PEI, though is transcribed at lower levels in most other mammary derived cultures. MGP requires vitamin K dependent gamma-carboxylation for its known function and thus can be inhibited by vitamin K antagonists. This raises the possibility that MGP may be among those factors that when inhibited by vitamin K antagonists reduce metastases in experimental models. Among the gene whose transcription is consistently suppressed upon mammary transformation were fibronectin and the type I keratin, K14. Differential cDNA screening therefore is an effective method of identifying genes involved in various aspects of mammary cell transformation. Francisco, California. SO - Oncogene 1990 Sep;5(9):1391-5. Neg-81. PMID:2505228 | PIR:A32794 TI - Cloning of cDNAs coding for human HMG I and HMG Y proteins: both are capable of binding to the octamer sequence motif. AB - In human B lymphocytes and placenta HMG I and its smaller isoform HMG Y are encoded by two distinct but structurally highly similar mRNAs which arise most likely by alternative splicing of a single primary transcript. Both have been cloned as cDNAs. On Northern blots an abundant mRNA species 2000 nucleotides in length was detected in all cell lines examined. Exclusively in erythroid cells an additional rare 3800 nucleotides long mRNA species was noted. In quiescent cells the mRNA levels of HMG I/Y were not significantly down-regulated. Southern blot analysis indicated that at least four genes are present per haploid human genome. Both proteins when expressed in bacteria bind specifically to A-T rich stretches of DNA suggesting that no posttranslational modifications are necessary for specific DNA binding. Interestingly, HMG I as well as HMG Y are capable of binding to the octamer transcriptional regulatory sequence motif. SO - Nucleic Acids Res 1989 Aug 11;17(15):5947-59. Neg-82. PMID:6322174 | PIR:IQECDK TI - Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. AB - The Escherichia coli dnaK gene is homologous to the major heat shock-induced gene in Drosophila (Hsp70). The primary DNA sequence of the entire protein-coding region of the dnaK gene was determined and compared with that of the Hsp70 gene of Drosophila. The two sequences are homologous; the dnaK gene could encode a 69,121-Da polypeptide, 48% identical to the hsp70 protein of Drosophila. The homology between the Hsp70 gene of Drosophila and the E. coli dnaK gene illustrates the remarkable conservation of the heat shock genes in evolution. In contrast to Drosophila and Saccharomyces cerevisiae, both of which contain multigene families related to the Hsp70 gene, hybridization analyses indicate that E. coli contains only a single Hsp70-related gene, dnaK. Hybridization between the DNA of an archaebacterium Methanosarcina barkeri and the Hsp70 genes of Drosophila, Saccharomyces, and E. coli has been detected, suggesting the existence of Hsp70-related genes in the three "primary kingdoms": eukaryotes, eubacteria, and archaebacteria. SO - Proc Natl Acad Sci U S A 1984 Feb;81(3):848-52. Neg-83. PMID:2394711 | PIR:GEHUM TI - Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene. AB - Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors. 92093. SO - J Biol Chem 1990 Sep 5;265(25):15040-8. Neg-84. PMID:1097438 | PIR:PEPG TI - Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin. AB - The complete amino acid sequence of porcine pepsin (EC 3.4.4.1) was constructed from the sequence of five cyanogen bromide fragments. The sequence of one of these fragments, CB2A, is reported here. The sequences of 4 other fragments are known from previous work. Porcine pepsin contains 327 residues with three structural variants. The active center aspartyl residue, which reacts with 1,2-epoxy-3-(p-nitrophenoxy)propane (Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247, 2566-2574), is located at residue 32. Another active site aspartyl residue, which reacts with diazo inactivators (Bayliss, R. S., Knowles, J. B., and Wybrandt, G. B. (1969) Biochem. J. 113, 377-386, IS LOCATED AT RESIDUE 215. The sequences around these 2 aspartyl residues are apparently homologous to each other. The sequences around the tryptophanyl residues at positions 39, 141, 181, and 300 are also homologous to one another. These homologous sequences could be genetic in origin. Fragment CB2A which contains 119 residues was constructed from the peptide sequences resulting from six proteolytic digestions and chemical cleavage at tryptophanyl bonds. SO - J Biol Chem 1975 Jul 10;250(13):5082-8. Neg-85. PMID:8397106 | PIR:A31780 TI - Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart. AB - Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M(r) isozyme. In B2 mRNA, exon 15 encodes four more residues than in B1. In B4 mRNA, exon 15 encodes six more residues than in B1, but exon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M(r) isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity. and Molecular Pathology, Brussels, Belgium. SO - FEBS Lett 1993 Sep 20;330(3):329-33. Neg-86. PMID:2768272 | PIR:ACRYD1 TI - The sidedness of the COOH terminus of the acetylcholine receptor delta subunit. AB - The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v ("43-kDa protein") as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular. Columbia University, New York, New York 10032. SO - J Biol Chem 1989 Sep 15;264(26):15457-63. Neg-87. PMID:8931545 | PIR:F2SPD2 FMSP32 TI - A model for the photosystem II reaction center core including the structure of the primary donor P680. AB - For a detailed understanding of the function of photosystem II (PSII), a molecular structure is needed. The crystal structure has not yet been determined, but the PSII reaction center proteins D1 and D2 show homology with the L and M subunits of the photosynthetic reaction center from purple bacteria. We have modeled important parts of the D1 and D2 proteins on the basis of the crystallographic structure of the reaction center from Rhodopseudomonas viridis. The model contains the central core of the PSII reaction center, including the protein regions for the transmembrane helices B, C, D, and E and loops B-C and C-D connecting the helices. In the model, four chlorophylls, two pheophytins, and the nonheme Fe2+ ion are included. We have applied techniques from computational chemistry that incorporate statistical data on side-chain rotameric states from known protein structure and that describe interactions within the model using an empirical potential energy function. The conformation of chlorophyll pigments in the model was optimized by using exciton interaction calculations in combination with potential energy calculations to find a solution that agrees with experimentally determined exciton interaction energies. The model is analyzed and compared with experimental results for the regions of P680, the redox active pheophytin, the acceptor side Fe2+, and the tyrosyl radicals TyrD and TyrZ. P680 is proposed to be a weakly coupled chlorophyll a pair which makes three hydrogen bonds with residues on the D1 and D2 proteins. In the model the redox-active pheophytin is hydrogen bonded to D1-Glu130 and possibly also to D1-Tyr126 and D1-Tyr147. TyrD is hydrogen bonded to D2-His190 and also interacts with D2-Gln165. TyrZ is bound in a hydrophilic environment which is partially constituted by D1-Gln165, D1-Asp170, D1-Glu189, and D1-His190. These polar residues are most likely involved in proton transfer from oxidized TyrZ or in metal binding. Stockholm University, Sweden. SO - Biochemistry 1996 Nov 19;35(46):14486-502. Neg-88. PMID:7691356 | PIR:A40303 TI - Cystic fibrosis transmembrane conductance regulator splice variants are not conserved and fail to produce chloride channels. AB - In the human CFTR only the rare exon 4- splice variant is conserved in mice. We have discovered two novel murine variants, exon 5- and exon 11b+. The exon 5- variant represents up to 40% of mRNA in all CFTR-expressing tissues and leaves the reading frame intact. The exon 11b+ variant inserts a novel exon between exons 11 and 12 with expression restricted to the testis. Two variants of 11b have been found and both introduce premature stop codons. When we expressed human CFTR variants lacking either exon 5 or exon 9 in HeLa cells, they failed to generate cAMP-mediated chloride transport, due to defective intracellular processing. The lack of conservation of splice variants between species and the inability of the more abundant splice variants to generate protein that is correctly processed argue against a physiological role and may simply represent aberrant splicing that is tolerated by the cell and organism. Brisbane, Australia. SO - Nat Genet 1993 Aug;4(4):426-31. Neg-89. PMID:6096808 | PIR:F2SP44 F2SPD2 TI - Structure of the spinach chloroplast genes for the D2 and 44 kd reaction-centre proteins of photosystem II and for tRNASer (UGA). AB - We have determined the sequence of the spinach (Spinacia oleracea) chloroplast genes for the photosystem II proteins, D2 and the 44 kd reaction-centre, chlorophyll a-binding protein, and for tRNASer (UGA). The 3' end of the D2 gene overlaps the first 50 bp of the 5' end of the gene for the 44 kd protein. Northern RNA hybridization analysis indicates the two genes are cotranscribed into a single 3.5 kb RNA. The predicted molecular weight of the 353-residue D2 protein is 39536 and that of the 473-residue 44 kd protein is 51816. Both proteins are hydrophobic containing at least five possible membrane-spanning domains. D2 shows significant homology to the 32 kd herbicide-binding protein (Zurawski et al., (1982) Proc. Natl. Acad. Sci. USA 79, 7699-7703), and parts of the 44 kd protein show obvious similarities to parts of the 51 kd reaction-centre, chlorophyll a-binding protein of photosystem II (Morris and Herrmann (1984) Nucleic Acids Res. 12, 2837-2850). The gene for tRNASer (UGA) which is on the opposite strand to and transcribed towards the photosystem II genes is 72% homologous with the corresponding Escherichia coli tRNASer. SO - Nucleic Acids Res 1984 Dec 11;12(23):8819-34. Neg-90. PMID:2744479 | PIR:A43803 TI - Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library. AB - Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA. SO - Gene 1989 Mar 15;76(1):171-5. Neg-91. PMID:6689612 | PIR:KKBOB TI - [Primary structure of cDNA of Bos taurus kappa-casein macropeptide] AB - By means of nucleotide sequencing in a library of clones, containing cDNA of Bos taurus mammary gland, a clone corresponding to kappa-casein has been identified. In addition, the region encoding for the complete nucleotide sequence of cDNA of kappa-casein B macropeptide has been detected. The nucleotide changes in the DNA sequence have been identified which mean that the isolated clone corresponds to the genetic variant B of kappa-casein. SO - Bioorg Khim 1983 Dec;9(12):1693-5. Neg-92. PMID:6897774 | PIR:KABOSB TI - Construction and identification by partial nucleotide sequence analysis of bovine casein and beta-lactoglobulin cDNA clones. AB - Double stranded (DS) DNA molecules obtained by reverse transcription of a partially purified lactating bovine mammary gland mRNA fraction were cloned into pBR322. Restriction maps for four recombinants were constructed and partial nucleotide sequence analysis of these revealed coding sequences corresponding to alpha s1-, beta-, and kappa-casein and beta-lactoglobulin. The specific single-stranded (SS) cDNAs representing each of these species were identified and their nucleotide lengths estimated. Evidence is presented that these are essentially full-length transcripts of the major mRNA species. On this basis, the cDNA clones range in size from 50% for beta-casein to about 95% for alpha s1-casein in comparison with their respective mRNAs. The DNA sequence spanning all eight phosphoserine residues in alpha s1-casein is presented. These data, together with other serine codon usage data, indicate that the mammary gland phosphoseryl tRNA does not play a role in the incorporation of serine phosphate residues during casein synthesis. The observation that the nucleotide sequences for the serine phosphate cluster in bovine alpha s1-and rat beta-casein exhibit close homology supports the suggestion that these regions have evolved from a common primordial sequence. SO - DNA 1982;1(4):375-86. Neg-93. PMID:2164224 | PIR:TPHUN1 TI - Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B. AB - The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) function as growth suppressors, we have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2. SO - Proc Natl Acad Sci U S A 1990 Jul;87(13):5148-52. Neg-94. PMID:3467321 | PIR:MMHUE4 TI - Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. AB - Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of non-erythroid tissues. In mammalian erythrocytes, it plays a key role in regulating membrane physical properties of mechanical stability and deformability by stabilizing spectrin-actin interaction. We report here the molecular cloning and characterization of human erythrocyte protein 4.1 cDNA and the complete amino acid sequence of the protein derived from the nucleotide sequence. Probes prepared from the cloned erythrocyte protein 4.1 cDNA hybridized with distinct mRNA species from a wide variety of non-erythroid tissues, including brain, liver, placenta, pancreas, and intestine, implying substantial homology between erythroid and non-erythroid protein 4.1. The availability of cloned erythrocyte protein 4.1 cDNA should facilitate the study of the functional characteristics of this protein in erythroid as well as non-erythroid cells. SO - Proc Natl Acad Sci U S A 1986 Dec;83(24):9512-6. Neg-95. PMID:2527334 | PIR:HHHU86 TI - Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein. AB - Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells. SO - Mol Cell Biol 1989 Jun;9(6):2615-26. Neg-96. PMID:2760048 | PIR:A33430 TI - Cloning and expression of a smooth muscle caldesmon. AB - Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data. 77030. SO - J Biol Chem 1989 Aug 15;264(23):13873-9. Neg-97. PMID:6303394 | PIR:C1HURB TI - Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r. AB - The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118. SO - Biochemistry 1983 Apr 12;22(8):1758-64. Neg-98. PMID:2262079 | PIR:GMDG TI - Cloning of canine gastrin cDNA's encoding variant amino acid sequences. AB - We have isolated 2 clones encoding gastrin from a canine antral mucosa cDNA library. The clones differed in nucleotide sequence at 2 sites in the coding region. One of the clones confirmed the amino acid sequence of dog gastrin obtained by peptide purification and analysis. An A-G substitution in the other clone resulted in a Thr-Ala substitution in the region encoding the biologically active carboxyl-terminal portion of gastrin. The source of the observed heterogeneity in cDNA sequences is not known but its location may have implications with regard to the physiology of gastrin in dogs. Ann Arbor. SO - Digestion 1990;46 Suppl 2:99-104. Neg-99. PMID:8336540 | PIR:A32541 TI - Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family. AB - Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences. University. SO - Mol Biol Evol 1993 May;10(3):497-511. Neg-100. PMID:2613684 | PIR:A33430 TI - Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin. AB - Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass. SO - J Biochem (Tokyo) 1989 Nov;106(5):778-83. Neg-101. PMID:2509275 | PIR:A33369 TI - Primary structure of rabbit skeletal muscle glycogen synthase deduced from cDNA clones. AB - The complete amino acid sequence of rabbit skeletal muscle glycogen synthase was deduced from cDNA clones with a composite length of 3317 bp. An mRNA of 3.6 kb was identified by Northern blot analysis of rabbit skeletal muscle RNA. The mRNA coded for a protein of 734 residues with a molecular weight of 83,480. The deduced NH2-terminal and COOH-terminal sequences corresponded to those reported for the purified protein, indicating the absence of any proteolytic processing. At the nucleotide level, the 5' untranslated and coding regions were 79 and 90% identical for rabbit and human muscle glycogen synthases, whereas the 3' untranslated regions were significantly less similar. The enzymes had 97% amino acid sequence identity. Interestingly, the NH2 and COOH termini of rabbit and human muscle glycogen synthase, the regions of phosphorylation, showed the greatest sequence variation (15 of 19 mismatches and two insertion/deletion events), which may indicate different evolutionary constraints in the regulatory and catalytic regions of the molecule. Indianapolis 46223. SO - FASEB J 1989 Nov;3(13):2532-6. Neg-102. PMID:1807167 | PIR:GEBOM GEHU TI - Direct identification of gamma-carboxyglutamic acid in the sequencing of vitamin K-dependent proteins. AB - We report the first direct method for the identification of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla), in the sequencing of proteins. The carboxyl groups on the protein are first converted to methyl esters with methanolic HCl, a procedure that reduces the polarity of the resulting ATZ derivative of dimethyl-Gla and so greatly improves its extraction from the polybrene-treated glass fiber filter. After conversion to the PTH derivative in methanolic HCl, the resulting dimethyl ester of PTH Gla can be identified directly by a simple modification of the standard HPLC program for the separation of PTH derivatives. This methylation procedure can be used to identify Gla residues in proteins bound to PVDF membranes, as we demonstrate for matrix Gla protein and prothrombin, and to evaluate directly the degree of partial gamma-carboxylation at given glutamic acid residues, as we demonstrate for the 50% gamma-carboxylation of residue 17 in human bone Gla protein. 92093. SO - Anal Biochem 1991 Nov 15;199(1):93-7. Neg-103. PMID:2447940 | PIR:SGHU1V TI - Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution. AB - The S-protein/vitronectin gene was isolated from a human genomic DNA library, and its sequence of about 5.3 kilobases including the adjacent 5' and 3' flanking regions was established. Alignment of the genomic DNA nucleotide sequence and the cDNA sequence indicated that the gene consisted of eight exons and seven introns. The intron positions in the S-protein gene and their phase type were compared to those in the hemopexin gene which shares amino acid sequence homologies with transin and the S-protein. Three introns have been found at equivalent positions; two other introns are very close to these positions and are interpreted as cases of intron sliding. Introns 3-7 occur at a conserved glycine residue within repeating peptide segments, whereas introns 1 and 2 are at the boundaries of the Somatomedin B domain of S-protein. The analysis of the exon structure in relation to repeating peptide motifs within the S-protein strongly suggests that it contains only seven repeats, one less than the hemopexin molecule. A very similar repeat pattern like that in hemopexin is shown to be present also in two other related proteins, transin and interstitial collagenase. An evolutionary model for the generation of the repeat pattern in the S-protein and the other members of this novel "pexin" gene family is proposed, and the sequence modifications for some of the repeats during divergent evolution are discussed in relation to known unique functional properties of hemopexin and S-protein. FRG. SO - Biochemistry 1987 Oct 20;26(21):6735-42. Neg-104. PMID:6409085 | PIR:UDCH TI - Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. AB - The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition. SO - Biochem J 1983 Apr 1;211(1):129-38. Neg-105. PMID:2769252 | PIR:HSBO2A HSBO3 TI - Predominant low-molecular-weight proteins in isolated brain capillaries are histones. AB - Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE. SO - J Neurochem 1989 Oct;53(4):1014-8. Neg-106. PMID:3877721 | PIR:GEBOM TI - Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. AB - The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation. SO - J Biol Chem 1985 Dec 5;260(28):14971-5. Neg-107. PMID:2280779 | PIR:INHUR TI - Structural and functional analysis of the insulin receptor promoter. AB - The insulin receptor plays a critical role in the maintenance of glucose homeostasis. Regulation of this key function must be under stringent controls. In order to study the regulation of insulin receptor gene expression, we have cloned, sequenced and characterized its promoter. The first exon of the insulin receptor gene is embedded in an unusual segment of DNA composed of Alu repeats. The promoter has the characteristics typical of a housekeeping gene. It is GC-rich and has multiple start sites of transcription. A 574 base pair fragment immediately upstream of the translation initiation site contains promoter activity when transfected into eukaryotic cell lines. Deletion analysis was performed to study promoter function. These studies showed that only 150 base pairs of promoter sequence were necessary for promoter function. This region contains three potential binding sites for the transcription factor, Sp1 and a TC box sequence. Furthermore, the fragment functions equally well in either orientation. We have defined an element in this region with enhancer function for both its homologous and a heterologous promoter. In addition, this region seems to contribute some degree of tissue specificity to insulin receptor gene expression. Diseases, National Institutes of Health, Bethesda, Maryland 20892. SO - Mol Endocrinol 1990 Apr;4(4):647-56. Neg-108. PMID:2833669 | PIR:KABOS2 KBBOA2 TI - Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species. AB - The nucleotide sequences corresponding to bovine alpha S2- and beta-casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk. Australia. SO - Mol Biol Evol 1987 May;4(3):231-41. Neg-109. PMID:3839187 | PIR:R6SSP2 R8SS12 TI - Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia. AB - The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein. SO - Eur J Biochem 1985 Jun 18;149(3):609-16. Neg-110. PMID:7488058 | PIR:FGHUA TI - In vivo degradation of human fibrinogen A alpha: detection of cleavage sites and release of antithrombotic peptides. AB - Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide extracts from human blood filtrate, a large number of fibrinogen A alpha fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen A alpha efficiently inhibits fibrinogen binding and platelet aggregation (IC50:20-50 microM) in vitro. We conclude that in vivo degradation of fibrinogen A alpha results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation. purification/pharmacology SO - Biochem Biophys Res Commun 1995 Oct 24;215(3):896-902. Neg-111. PMID:728424 | PIR:PHRBG TI - Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control. AB - The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits. SO - Biochemistry 1978 Dec 26;17(26):5657-72. Neg-112. PMID:728425 | PIR:PHRBG TI - Amino acid sequence of two cyanogen bromide fragments of glycogen phosphorylase. AB - This communication presents the strategy and experimental details to prove the amino acid sequence of two large fragments of rabbit muscle glycogen phosphorylase generated by cleavage with cyanogen bromide. These fragments, CB18 and CB15, represent 241 of the 841 residues in the whole molecule. In addition to applying methods of automated liquid phase Edman degradation, techniques of selective immobilization and solid phase Edman degradation are used. One of the two cyanogen bromide fragments (CB15) contains two of the sites of cleavage with hydroxylamine which have proved to be important in the overall strategy of determining the complete sequence of this molecule. Together with the accompanying reports by Koide, A., et al., and Titani, K., et al. ((1978) Biochemistry 17 (first and third papers, respectively, in a series in this issue)), the present communication completes the proof of the amino acid sequence of phosphorylase and provides the basis for examining the relationship between its structure and function. SO - Biochemistry 1978 Dec 26;17(26):5672-9. Neg-113. PMID:728426 | PIR:PHRBG TI - Sequence of the carboxyl-terminal 492 residues of rabbit muscle glycogen phosphorylase including the pyridoxal 5'-phosphate binding site. AB - This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined. SO - Biochemistry 1978 Dec 26;17(26):5680-93. Neg-114. PMID:3426601 | PIR:B32541 SBHUP TI - Human submandibular gland statherin and basic histidine-rich peptide are encoded by highly abundant mRNA's derived from a common ancestral sequence. AB - The molecular cloning of sequences encoding human submandibular gland (SMG) statherin and a basic histidine-rich peptide is described. The corresponding mRNA's were highly abundant in the human and the Rhesus monkey (Macaca mulatta) SMG, but no homologous message was detectable in the murine SMG. Sequence analysis revealed strong homology between the statherin and basic histidine-rich mRNA's, suggesting their evolution from a common ancestral sequence. York, Buffalo 14214. SO - Biochem Biophys Res Commun 1987 Dec 16;149(2):784-90. Neg-115. PMID:2223773 | PIR:A35702 TI - Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor. AB - Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin. SO - Biochemistry 1990 Aug 14;29(32):7414-20. Neg-116. PMID:1942038 | PIR:OACH TI - Crystal structure of uncleaved ovalbumin at 1.95 A resolution. AB - Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed. SO - J Mol Biol 1991 Oct 5;221(3):941-59. Neg-117. PMID:1709163 | PIR:DVHUCF TI - The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin. AB - Evaluation of the expression of the cystic fibrosis (CF) gene in human epithelial cell lines demonstrated active, but low level, transcription of the gene. Analysis of 3.8 kilobases of genomic sequences 5' to exon 1 of the CF gene demonstrated no TATA promoter element, but a high G + C content, multiple transcription start sites, and several potential Sp1 binding sites. Fragments of 5'-flanking sequences from 2.2 kilobases to as small as 102 base pairs 5' to the major transcription start site supported constitutive reporter gene expression in epithelial cells, but at low levels, and independent of the length of the 5' fragment. CF gene transcription was down-regulated by phorbol myristate acetate. Importantly, evaluation of freshly isolated normal human bronchial cells also demonstrated CF gene transcription at a relatively low rate. Together, these observations suggest that although the normal CF gene promoter has characteristics of a "housekeeping"-type gene, and the gene is expressed at low levels in cells of organs that manifest the clinical disorder "cystic fibrosis," its expression can be modulated transcriptionally, implying a possible therapeutic approach for the disease. Institutes of Health, Bethesda, Maryland 20892. SO - J Biol Chem 1991 May 15;266(14):9140-4. Neg-118. PMID:8130199 | PIR:ACRYD1 TI - Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications. AB - To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet. 02115. SO - Biochemistry 1994 Mar 15;33(10):2859-72. Neg-119. PMID:1299613 | PIR:KABOSB TI - Isolation of a new ligand-carrying casein fragment from bovine mammary gland microsomes. AB - Whilst looking for components involved in retinol metabolism in secreting mammary gland cells, a 12 kDa protein was isolated. This protein had bound a ligand with characteristics of retinol. N-Terminal sequencing and amino acid analysis showed that this protein is highly homologous with an alpha-s1-casein fragment. No ligand was found for beta-lactoglobulin, previously thought to be involved in retinol metabolism. SO - FEBS Lett 1992 Jul 6;305(3):189-91. Neg-120. PMID:2373369 | PIR:SBHUP TI - Structure and sequence determination of the gene encoding human salivary statherin. AB - Human statherin (STT) is a low-Mr (43 amino acids) acidic phosphoprotein secreted mainly by salivary glands. It acts as an inhibitor of precipitation of Ca.phosphate salts in the oral cavity. DNA (12.2 kb) was isolated from human genomic phage lambda libraries as a series of overlapping clones, and the nucleotide sequence of the STT-encoding gene (STT) was determined. The transcribed region spans 6.5 kb and contains six exons and five introns. Upstream DNA (1.6 kb) was also sequenced and a number of possible regulatory elements were identified. The exon-intron boundaries of the STT gene roughly coincide with the protein-coding regions of the mRNA and with the functional domains of STT. This pattern of organization has been seen in a variety of eukaryotic genes and is consistent with the domain theory of gene evolution. SO - Gene 1990 May 14;89(2):245-51. Neg-121. PMID:6274862 | PIR:OKBOG TI - Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase. AB - The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile- Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-L ys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins. SO - J Biol Chem 1982 Jan 25;257(2):727-33. Neg-122. PMID:7588731 | PIR:HHHU86 TI - Mechanism of dimer formation of the 90-kDa heat-shock protein. AB - This study describes the mechanism of homodimer formation of the 90-kDa heat-shock protein (HSP90). In eukaryotic cells, there are two HSP90 isoforms, alpha and beta, encoded by two separate genes. HSP90 alpha exists predominantly as a homodimer, HSP90 beta mainly as a monomer. Analysis by native PAGE revealed that bacterially expressed HSP90 alpha fused to glutathione S-transferase (GST) existed as a high-molecular-mass oligomer, and was converted to a homodimer following removal of the fusion enzyme by thrombin cleavage. A deletion mutant, HSP90 alpha D44-603, formed a monomer and an N-terminal truncated mutant, HSP90 alpha 533-732, existed as a dimer, indicating that the dimer-forming ability resides somewhere in the C-terminal 200 amino acids. Limited proteolysis of the C-terminal 200 amino acids of HSP90 alpha with chymotrypsin produced the C-terminal 16-kDa fragment (Met628/Ala629-Asp732) and its adjacent more N-terminal 13-kDa fragment (Val542-Tyr627/Met628). Size-exclusion HPLC and two-dimensional PAGE analyses demonstrated that these two chymotryptic fragments bound each other. The C-terminal 198 amino acids as well as the full-length form of HSP90 beta revealed a lower dimer-forming activity than HSP90 alpha. Expression of the chimeric proteins at the C-terminal 198 amino acids of the alpha and beta isoforms further indicated that the 16 amino acid substitutions locating between amino acids 561 and 685 account for the impeded dimerization of HSP90 beta. A leucine zipper motif (Met402-Leu423) was unlikely to be involved in the dimer formation. Taken together, these results indicate that the dimeric structure of HSP90 alpha is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met628/Ala629-Asp732) of one subunit and the adjacent more N-terminal region (Val542-Try627/Met628) of the other subunit. Morioka, Japan. SO - Eur J Biochem 1995 Oct 1;233(1):1-8. Neg-123. PMID:670209 | PIR:PHRBG TI - Inactivation of phosphorylase b by potassium ferrate. Identification of a tyrosine residue involved in the binding of adenosine 5'-monophosphate. AB - The site of reaction of potassium ferrate (K2FeO4) with rabbit muscle phosphorylase b has been further characterized in an extension of previously published studies (Lee, Y. M., and Benisek, W. F. (1976) J. Biol, Chem. 251, 1553-1560) reporting inactivation of the enzyme by this reagent. The tryptic peptide composed of residues 70 to 80 of the enzyme's polypeptide chain was shown to contain a tyrosine residue which is chemically modified by ferrate and which is protected by 5'-AMP. The sequence of this peptide obtained from both untreated and ferrate-treated phosphorylase b was determined, and the results showed that tyrosine-75 was the residue with which ferrate reacts. SO - J Biol Chem 1978 Aug 10;253(15):5460-3. Neg-124. PMID:2498649 | PIR:QRBOT1 TI - Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. AB - Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol. SO - Mol Cell Biol 1989 Apr;9(4):1381-8. Neg-125. PMID:398910 | PIR:R3RTS6 TI - Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a, L18, L27, L30, L37, L37a, and L39. AB - The sequence of the amino-terminal region of eleven rat liver ribosomal proteins--S4, S6, S8, L6, L7a, L18, L27, L30, L37a, and L39--was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry. SO - J Supramol Struct 1979;12(4):425-33. Neg-126. PMID:1986798 | PIR:EQBOA TI - Tissue kallikrein processes small proenkephalin peptides. AB - Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met5-enkephalin and Met5-Lys6-enkephalin. Met5-Lys6-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37 degrees C and pH 8.5, the KM values for formation of Met5-enkephalin and Met5-Lys6-enkephalin were 129 and 191 microM, respectively. Corresponding kcat values were 0.001 and 0.03 s-1 and kcat/KM ratios were 8 and 1.6.10(2) M-1.s-1, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met5-Arg6-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, KM was 64 microM, kcat was 4.5 s-1, and the kcat/KM ratio was 7.10(4) M-1.s-1. At 5.5, the pH of the secretory granules, KM, kcat and kcat/KM were 184 microM, 1.9 s-1 and 10(4) M-1.s-1, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds. SO - Biochim Biophys Acta 1991 Jan 8;1076(1):9-14. Neg-127. PMID:7999787 | PIR:S52083 TI - Characterization of a bovine mammary gland PP3 cDNA reveals homology with mouse and rat adhesion molecule GlyCAM-1. AB - A full length PP3 (Proteose-Peptone component 3) cDNA of 679 bp was isolated from a bovine mammary gland cDNA library. The cDNA encodes a signal peptide of 18 amino acids followed by the mature PP3 sequence of 135 amino acids. This polypeptide showed homology with mouse and rat GlyCAM-1 (Glycosylation dependent Cell Adhesion Molecule 1) a protein which has been shown to act as a ligand for lymphocytes. The similarity was most profound between the signal peptides and three short regions of the mature polypeptides. Additionally structural conservation was predicted by computer analysis in the shape of a C-terminal amphipathic helix. PP3 was found to be expressed in mammary gland but not in peripheral lymph nodes, Peyer's pathes, lung, spleen, heart, and muscle. SO - Biochim Biophys Acta 1995 Jan 2;1260(1):116-8. Neg-128. PMID:2538124 | PIR:INHUR TI - Alternative splicing of human insulin receptor messenger RNA. AB - The polymerase chain reaction has been used to examine alternative splicing of human insulin receptor (hINSR) mRNA. Alternative splicing of a 36 base pair exon, exon 11, generates hINSR transcripts encoding receptor isoforms which differ in sequence at the C-terminal end of the insulin-binding alpha-subunit. This process appears to be tissue-specific and, in addition, may be developmentally regulated. Chicago, IL 60637. SO - Biochem Biophys Res Commun 1989 Feb 28;159(1):312-6. Neg-129. PMID:1582406 | PIR:I38344 TI - Towards a molecular understanding of titin. AB - Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M- to Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A-band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M-line encode a kinase domain and are closely related to the C-terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A-band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin-C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super-repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C-protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A-band titin is likely to be involved in the ordered assembly of the vertebrate thick filament. SO - EMBO J 1992 May;11(5):1711-6. Neg-130. PMID:2415509 | PIR:A24608 A30142 B30142 CMBO PEHU PEPG TI - Isolation of human, swine, and rat prepepsinogens and calf preprochymosin, and determination of the primary structures of their NH2-terminal signal sequences. AB - The total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43,000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows: (Sequence: see text). These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase. SO - J Biochem (Tokyo) 1985 Aug;98(2):483-92. Neg-131. PMID:6091741 | PIR:OKBOG TI - Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. AB - The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme. SO - Biochemistry 1984 Aug 28;23(18):4207-18. Neg-132. PMID:2730665 | PIR:A33430 TI - 35 kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T. AB - Using a tropomyosin-coupled affinity column, we have demonstrated a direct association between the chymotryptic 35 kDa fragment of h-caldesmon, which is located at the C-terminal of the parent molecule, and gizzard tropomyosin. We have subsequently determined the nucleotide sequence of cDNA clones encoding the 35 kDa fragment from the cDNA library prepared from chick embryo gizzards, and have deduced the amino acid sequence. Calculating from the predicted sequence, the 35 kDa fragment is composed of 306 amino acid residues. In agreement with the tropomyosin-binding ability, the 35 kDa fragment conserves two consensus sequences of the tropomyosin-binding domain in troponin T. These results suggest that the 35 kDa fragment of h-caldesmon, at least in part, has a common property to the striated muscle troponin T. Medical School, Japan. SO - Biochem Biophys Res Commun 1989 May 30;161(1):38-45. Neg-133. PMID:8143716 | PIR:ACRYD1 TI - All potential glycosylation sites of the nicotinic acetylcholine receptor delta subunit from Torpedo californica are utilized. AB - All possible N-glycosylation sites of the delta subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high-mannose-type oligosaccharide is bound at Asn143 of the delta subunit. The oligosaccharides at positions Asn70 and Asn208 of the delta subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit. SO - Eur J Biochem 1994 Mar 15;220(3):1005-11. Neg-134. PMID:2783136 | PIR:FGHUA TI - The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation. AB - The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation. UK. Acid) SO - Biochem Biophys Res Commun 1989 Sep 29;163(3):1256-64. Neg-135. PMID:2546149 | PIR:TPHUN1 TI - Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. AB - The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists of a single chain of 321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-like molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence similarities. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr). SO - Proc Natl Acad Sci U S A 1989 Jul;86(14):5252-6. Neg-136. PMID:3286634 | PIR:A32541 B32541 TI - Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. AB - Histatins 1, 3, and 5 from human parotid secretion were isolated by gel filtration on Bio-Gel P-2 and reverse phase high performance liquid chromatography. The complete amino acid sequences of histatins determined by automated Edman degradation of the proteins, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows: (Sequence: see text). Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues, have molecular weights of 4929, 4063, and 3037, respectively, and contain 7 residues of histidine. Histatin 1 contains 1 mol of phosphate/mol of protein; histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of all three histatins are identical, and the carboxyl-terminal 7 residues of histatins 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly- (residues 23-29), in histatin 1 is absent in histatin 3; and the sequence, -Gly-Tyr-Arg- (residues 23-25), in histatin 3 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino terminal 24 residues of histatin 3. The structural data suggest that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. All three histatins exhibit the ability to kill the pathogenic yeast, Candida albicans. Massachusetts. SO - J Biol Chem 1988 Jun 5;263(16):7472-7. Neg-137. PMID:2566545 | PIR:INHUR TI - Molecular and clinical characterization of an insertional polymorphism of the insulin-receptor gene. AB - A restriction-fragment-length polymorphism (RFLP) detected with the human insulin-receptor cDNA and the enzyme Sac I has been reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM) in White and Black populations and segregated with diabetes in two small pedigrees with maturity-onset diabetes of the young. A size difference of approximately 500 base pairs (bp) was demonstrated between the alleles of this and several other RFLPs that mapped to the same 300-bp region near the transmembrane coding region of the cDNA beta-chain, thus suggesting the presence of an insertion in this region that could affect insulin-receptor function. Genomic DNA fragments containing this RFLP were cloned from an individual heterozygous for the putative insertion, and the differing fragments of the two alleles were sequenced. The presence of a 400-bp insertion was thus confirmed and was demonstrated to be entirely within an intron. No significant coding-region differences from published cDNA sequences were detected in four exons sequenced from the region of the insertional allele. The sequenced regions included multiple Alu repeat sequences. The RFLP was unusual in that the larger allele consisted of an additional Alu repeat sequence that included a new Pst I site. Because the nature and location of the insertion did not suggest a role in insulin-receptor function, the association of this RFLP with NIDDM and hyperinsulinemia was reexamined in a small sample of Whites. No association could be demonstrated, and the insertion also failed to segregate with NIDDM in five White pedigrees.(ABSTRACT TRUNCATED AT 250 WORDS) Lake City, Utah. SO - Diabetes 1989 Jun;38(6):737-43. Neg-138. PMID:8513794 | PIR:HSHUP2 TI - Amino acid sequence of the human intermediate basic protein 2 (HPI2) from sperm nuclei. Structural relationship with protamine P2. AB - Human intermediate basic protein 2 (HPI2) is a low-molecular-mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N-terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys-C and Glu-C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N-terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues. The amino acid sequence of HPI2 (81 residues) is identical to the sequence of the C-terminal region of another minor sperm nuclear protein, human intermediate basic protein 1 (HPI1, 101 residues), which was sequenced previously [Martinage, A., Arkhis, A., Alimi, E., Sautiere, P. & Chevaillier, P. (1990) Eur. J. Biochem. 191, 449-451]. Due to this structural similarity, HPI2 must be considered as an intermediate in the maturation of proprotamine HPI1 limited proteolysis. Creteil, France. SO - Eur J Biochem 1993 Jun 1;214(2):445-50. Neg-139. PMID:1860879 | PIR:S67669 TI - Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression. AB - Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% identical in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport. Republic of Germany. SO - J Cell Biol 1991 Aug;114(3):443-53. Neg-140. PMID:272676 | PIR:OACH TI - Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. AB - Ovalbumin mRNA was translated in a reticulocyte lysate. The primary translation product starts with methionine derived from Met-tRNAf. When the nascent polypeptide is about 20 residues long, this methionine is removed. The new NH2-terminal glycine is acetylated from acetyl-CoA when the polypeptide is 44 residues long. The sequence of 35 residues at the NH2 terminus of ovalbumin was determined by automated Edman degradation after a method was devised to prevent acetylation during protein synthesis in the reticulocyte lysate. This sequence is the same as that of secreted ovalbumin and does not resemble the transient "signal peptides" associated with most secretory proteins, including three other egg white proteins synthesized in the same cells as ovalbumin. SO - Proc Natl Acad Sci U S A 1978 Jan;75(1):94-8. Neg-141. PMID:2211730 | PIR:INHUR TI - Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol. AB - Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds. Genetics, Duarte, California. SO - J Biol Chem 1990 Oct 25;265(30):18673-81. Neg-142. PMID:3531202 | PIR:MMHUE4 TI - Structure of the spectrin-actin binding site of erythrocyte protein 4.1. AB - The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily. SO - J Biol Chem 1986 Oct 5;261(28):13362-6. Neg-143. PMID:3131325 | PIR:QRBOT1 QRBOT2 TI - Microtubule-binding domain of tau proteins. AB - Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain. of Tokyo, Japan. SO - J Biol Chem 1988 Jun 5;263(16):7703-7. Neg-144. PMID:1180911 | PIR:TPRBIS TI - The amino acid sequence of troponin I from rabbit skeletal muscle. AB - The complete amino acid sequence of rabbit skeletal muscle troponin I was determined by the isolation of the cyanogen bromide fragments and the tryptic methionine-containing peptides. Troponin I contains 179 amino acid residues and has a molecular weight of 20864. Its N-terminus is acetylated. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50055 (23 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5. SO - Biochem J 1975 Aug;149(2):493-6. Neg-145. PMID:1420367 | PIR:OKBO2C TI - Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase. AB - The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues. Heidelberg, Germany. SO - Biochim Biophys Acta 1992 Nov 15;1171(1):93-6. Neg-146. PMID:2760073 | PIR:A40936 TI - Molecular cloning of a novel human leukemia-associated gene. Evidence of conservation in animal species. AB - We have recently described an 18-kilodalton polypeptide (p18) that is present in much greater abundance in acute leukemic blast cells (myeloid and lymphoid) than in resting or proliferating nonleukemic lymphoid cells or chronic lymphoid and myeloid leukemic cells. In this report we describe the cloning of two different sized full-length cDNAs that code for p18. The two cDNAs differ in their 3'-noncoding regions as a result of alternative polyadenylation. Analysis of the complete nucleotide sequence and the corresponding amino acid sequence did not reveal significant homology to any previously described sequences. We show evidence that this gene is highly conserved in several animal species and low stringency hybridization studies suggest that the p18 gene may be a member of a family of partially homologous genes in the human genome. 48109. SO - J Biol Chem 1989 Aug 25;264(24):14556-60. Neg-147. PMID:2781520 | PIR:TIHUGK TI - Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein. AB - Lipoprotein-Associated Coagulation Inhibitor (LACI) is a factor Xa dependent inhibitor of the factor VII(a)/Tissue Factor catalytic complex. Deduced from partial cDNA sequence, LACI's amino acid sequence has recently been reported. Northern blot analysis showed LACI cDNA hybridizes to RNAs of 1.4 and 4.0 kb in size. To complete the characterization of the LACI message(s), overlapping LACI cDNAs were isolated from a human endothelial cell library. Sequence analysis revealed the clones' inserts span 4023 bases of sequence, consisting of 381 bases of 5' untranslated sequence, an open reading frame of 912 bases, 2682 bases of 3' untranslated sequence and 48 bases of poly(A) sequence. In addition, a short 1.4 kb insert which encodes for LACI was found to contain 49 bases of 3' untranslated sequence and a 3' poly(A) tail. The 1.4 kb of sequence is contained in the 4.0 kb sequence, except for 14 bases of 5' sequence, suggesting that the LACI messages arise by the use of alternative termination and polyadenylation signals during processing. Northern blot analysis of RNA isolated from cells treated with actinomycin D showed both RNA species appear to be relatively stable. Using a bovine papilloma virus vector, LACI cDNA was transfected into mouse C127 fibroblasts. The recombinant LACI is recognized by polyclonal anti-LACI IgG, binds to factor Xa and inhibits VII(a)/Tissue Factor activity in a similar fashion as LACI purified from HepG2 cell conditioned media. St. Louis, Missouri. purification SO - Thromb Res 1989 Jul 1;55(1):37-50. Neg-148. PMID:2841317 | PIR:KIZMPO TI - Primary structure of maize pyruvate, orthophosphate dikinase as deduced from cDNA sequence. AB - We have isolated two overlapping cDNA clones that encompass the entire structural gene for pyruvate, orthophosphate dikinase from maize. The analysis of the nucleotide sequence has revealed that the cDNA clones include an insert of a total of 3,171 nucleotides without a poly(A) tail and encode a polypeptide that contains 947 amino acid residues and has a molecular weight of 102,673. Comparison of the N-terminal amino acid sequence of purified pyruvate, orthophosphate dikinase protein with that deduced from the nucleotide sequence shows that the mature form of pyruvate, orthophosphate dikinase in the maize chloroplast consists of 876 amino acid residues and has a molecular weight of 95,353. The amino acid composition of the deduced sequence of pyruvate, orthophosphate dikinase is in good agreement with that of the purified enzyme. The region that contains the active and regulatory sites of pyruvate, orthophosphate dikinase can be found in the deduced sequence of amino acids. We have predicted the secondary structure and calculated the hydropathy pattern of this region. The extra 71 residues at the N terminus of the deduced sequence of amino acid residues corresponds to the transit peptide which is indispensable for the transport of the precursor protein into chloroplasts. We have compared the primary structure of the pyruvate, orthophosphate dikinase transit peptide to those of other proteins and found sequences similar to the consensus sequences found in other transit peptides. SO - J Biol Chem 1988 Aug 15;263(23):11080-3. Neg-149. PMID:6304716 | PIR:VEHY TI - Primary and secondary structure of hamster vimentin predicted from the nucleotide sequence. AB - The nucleotide sequence of two recombinant plasmids containing hamster vimentin cDNA was determined. The sequence comprises 1,640 base pairs and reveals virtually the total primary structure of vimentin and a large part of the 3' noncoding region. Secondary structure prediction methods allow the characterization of two distinct regions of the polypeptide chain, 135 and 145 residues long, which are able to form alpha helices organized in "coiled coils." Three nonhelical domains can be distinguished: a very basic NH2-terminal domain of at least 67 residues, a nonhelical region of 45 amino acids separating the two helix domains, and a COOH-terminal region of 55 residues, which contains an excess of acidic amino acids. The meaning of each of these domains of the vimentin polypeptide for the subunit and filament formation is discussed. SO - Proc Natl Acad Sci U S A 1983 Jun;80(12):3548-52. Neg-150. PMID:3527226 | PIR:HSHUP1 HSHUP2 TI - Isolation and amino-acid sequence analysis of human sperm protamines P1 and P2. Occurrence of two forms of protamine P2. AB - The two protamines of human sperm cell nuclei, P1 and P2, were isolated in pure form after extraction with 6M guanidine/5% mercaptoethanol and alkylation with vinyl pyridine by reversed-phase high-performance liquid chromatography. The amino-acid sequence of protamine P1 was determined by analysing the intact protein and the fragments obtained by cyanogen bromide cleavage. Out of the 50 amino-acid residues 24 are arginines and 6 are cysteines. The sequence of protamine P2 was determined by analysing the intact protein and the fragments resulting from cleavage with endoproteinase Lys-C and thermolysin. Protamine P2 was found to occur in two forms which only differ in their N-terminal regions. The form P2' is three amino-acid residues longer at the N-terminus than the form P2''. Out of the 57 amino-acid residues in the longer form 27 are arginines and 5 are cysteines. Human protamine P1 is highly homologous with the protamines isolated from bull, boar, ram and mouse sperm cells, but human protamine P2 shows a novel type of structure, although also here the dominant amino acids are arginine and cysteine. SO - Biol Chem Hoppe Seyler 1986 Jun;367(6):515-22. Neg-151. PMID:2358074 | PIR:A40936 TI - A single amino acid difference distinguishes the human and the rat sequences of stathmin, a ubiquitous intracellular phosphoprotein associated with cell regulations. AB - Stathmin is a ubiquitous phosphoprotein proposed to play a general role as an intracellular relay integrating diverse regulatory signals of the cell's environment. We used a rat stathmin probe to isolate two classes of cDNAs coding for the human protein and corresponding to the usage of different polyadenylation sites. Compared to the rat sequences, they displayed a very high conservation both at the nucleic acid and the deduced protein sequence levels, with a single conservative amino acid difference. Further analysis of the protein sequence revealed novel putative phosphorylation sites, as well as internal repeated sequences which might reflect structural features involved in the molecular mechanisms by which stathmin fulfills its biological functions. The extreme conservation of the entire stathmin sequence further stresses the essential and general role of stathmin in cell regulations. SO - FEBS Lett 1990 May 21;264(2):275-8. Neg-152. PMID:2475911 | PIR:DVHUCF TI - Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. AB - Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients. Canada. SO - Science 1989 Sep 8;245(4922):1066-73. Neg-153. PMID:1377165 | PIR:A39901 TI - Expression and chromosome localization of the murine cystic fibrosis transmembrane conductance regulator. AB - A 13.5-kb genomic fragment of the mouse cystic fibrosis transmembrane conductance regulator (CFTR) gene was isolated from a C57BL/6J liver DNA library, using a human CFTR exon 10 probe. This region of the human gene includes the most common cystic fibrosis mutation (deletion of the Phe508 residue) in the first nucleotide binding domain of CFTR. Sequence analysis demonstrated 87% identity between the predicted mouse and the normal human CFTR exon 10 sequences, including conservation of the Phe508 residue. Northern analysis revealed that the mouse gene is expressed in intestine, lung, stomach, kidney, and salivary gland. In contrast to human CFTR, murine CFTR transcripts were not detectable by Northern analysis in the liver or pancreas. More sensitive PCR analysis, however, revealed that the mouse CFTR gene is weakly expressed in other tissues, including liver and pancreas. During development, mouse CFTR transcripts were observed as early as Embryonic Day 13. Southern analysis of mouse x Chinese hamster somatic cell hybrid DNAs mapped the mouse CFTR locus (Cftr) to Chromosome 6 (Chr 6). Subsequent typing of the progeny of an interspecies backcross revealed that Cftr is closely linked to the proto-oncogene c-met locus (Met) in the centromeric region of mouse Chr 6, consistent with the observation that there is a conserved chromosomal segment on human chromosome 7 and mouse Chr 6. Neurobiology, New York, New York 10029. SO - Genomics 1992 Jun;13(2):381-8. Neg-154. PMID:2591742 | PIR:HHHU86 TI - Cloning and analysis of a human 86-kDa heat-shock-protein-encoding gene. AB - An 86-kDa heat-shock-protein-encoding (hsp86) cDNA probe permitted to identify, in whole genomic human DNA, two EcoRI fragments of 2.6 and 5.3 kb. These two fragments, as well as an homologous phage lambda VIII1 harboring about 19 kb of human DNA, were isolated from genomic libraries. Sequence analysis revealed that three different genomic hsp86 sequences had been cloned, one of them being the 5' half of a functional gene. This gene contains several introns, as compared to the entire Hsp86-encoding sequence found in lambda VIII1, which represents a processed pseudogene. Cloned hsp86 promoter, with its TATA-box and a heat-shock element upstream at nt positions -25 and -75, respectively, was functional, as verified by fusion to the bacterial chloramphenicol acetyltransferase-encoding gene and its transient expression in vivo. The typical hsp86-type heat-shock regulation was observed, i.e., significant basal activity associated with an inducibility at elevated temperatures. Furthermore, accurate and efficient in vitro transcription was initiated at this hsp86 promoter, resulting in expression of the hsp86 gene, as well as the unrelated sequences. SO - Gene 1989 Nov 15;83(1):105-15. Neg-155. PMID:9278503 | PIR:A29234 A32412 A38742 A39129 A39292 A42863 A42959 A43325 A55345 ADEC2A ADECFP ADECOG AJECDS AJECN AJECQ APECA AYEC B41856 B49749 B65056 BKEC9 BLEC BLECGP BYEC BYECPR C64736 C64901 C64989 C65131 CBEC62 D35905 D64775 D64925 DCECDM DCECIS DEECFS DEECM DEECTH DPECM DSECF DSECN DTECR DWECHD DWECS DWECTD E64946 E65073 EFECG EFECT EFECTA F64828 F65135 FIEC1 FIEC2 FIEC3 GBEC GDEC GREC H64764 H64965 H64983 I41215 IBEC IQECDK JC2265 JDEC22 JGECG JGECM JGECR JH0242 JKECQ JN0289 JQ1149 JS0628 JS0662 JT0742 JU0300 KIECEG KIECGL KIECRY KMECPW LPECW LWECA MMECF MMECZB NRECD OWECF OXECLD PAECA Q4ECFR QKEC QQECW1 QREC3M QRECC QRECCS QRECCY QRECCZ QRECIC QRECS R3EC1 R3EC10 R3EC11 R3EC12 R3EC13 R3EC14 R3EC15 R3EC16 R3EC17 R3EC18 R3EC19 R3EC2 R3EC20 R3EC21 R3EC3 R3EC4 R3EC5 R3EC6 R3EC7K R3EC8 R3EC9 R5EC1 R5EC10 R5EC11 R5EC13 R5EC14 R5EC15 R5EC16 R5EC17 R5EC18 R5EC19 R5EC2 R5EC20 R5EC21 R5EC22 R5EC23 R5EC24 R5EC25 R5EC27 R5EC28 R5EC29 R5EC3 R5EC30 R5EC31 R5EC32 R5EC33 R5EC34 R5EC35 R5EC36 R5EC4 R5EC5 R5EC6 R5EC7 R5EC9 RDEC1R RDEC2R RDECFF RDECFS RDECPA RDECSH RDECT RGECLR RGECP2 S00252 S00903 S01788 S01789 S03785 S04782 S05491 S08345 S08623 S15181 S15945 S20460 S22662 S23807 S30268 S30269 S31883 S41589 S56397 S56594 S56606 S57828 S70788 S78604 SNECPI SYECBB SYECCG SYECDP SYECKR SYECMT SYECPF SYECSA SYECSM SYECTP SYECZ1 TSECB TXEC UFECDW WQEC2M WQECPH WQECPI XDEC XNECD XNECV XUECGA XXECDP XXECTG XYECMH YNEC YUEC YXECUG ZPECS TI - The complete genome sequence of Escherichia coli K-12. AB - The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer. Madison, WI 53706, USA. ecoli@genetics.wisc.edu SO - Science 1997 Sep 5;277(5331):1453-74. Neg-156. PMID:8414980 | PIR:A32794 TI - Organization, inducible-expression and chromosome localization of the human HMG-I(Y) nonhistone protein gene. AB - Members of the HMG-I(Y) family of mammalian nonhistone proteins are of importance because they have been demonstrated to bind specifically to the minor groove of A.T-rich sequences both in vitro and in vivo and to function as gene transcriptional regulatory proteins in vivo. Here we report the cloning, sequencing, characterization and chromosomal localization of the human HMG-I(Y) gene. The gene has several potential promoter/enhancer regions, a number of different transcription start sites and numerous alternatively spliced exons making it one of the most complex nonhistone chromatin protein-encoding genes so far reported. The putative promoter/enhancer regions each contain a number of conserved nucleotide sequences for potential binding of inducible regulatory transcription factors. Consistent with the presence of these conserved sequences, we found that transcription of the HMG-I(Y) gene is inducible in human lymphoid cells by factors such as phorbol esters and calcium ionophores. Detailed sequence analysis confirms our earlier suggestion that alternative splicing of precursor mRNAs gives rise to the major HMG-I and HMG-Y isoform proteins found in human cells. Furthermore, the gene's exon-intron arrangement fully accounts for all of the previously cloned human HMG-I(Y) cDNAs (1,2). Also of considerable interest is the fact that each of the three different DNA-binding domain peptides present in an individual HMG-I(Y) protein is coded for by sequences present on separate exons thus potentially allowing for exon 'shuffling' of these functional domains during evolution. And, finally, we localized the gene to the short arm of chromosome 6 (6p) in a region that is known to be involved in rearrangements, translocations and other abnormalities correlated with a number of human cancers. Pullman 99164-4660. SO - Nucleic Acids Res 1993 Sep 11;21(18):4259-67. Neg-157. PMID:2857492 | PIR:WHRTY TI - Complete coding sequence of rat tyrosine hydroxylase mRNA. AB - Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed. SO - Proc Natl Acad Sci U S A 1985 Jan;82(2):617-21. Neg-158. PMID:1396676 | PIR:IQECDK XNCHDM TI - Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK. AB - On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70). Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated. The complex dissociated on treatment with MgATP or at pH 5.5. Like the mature enzyme, pmAspAT is a dimer (2 x 47 kDa) and exhibits about a third of its enzyme activity. In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer. The complex is catalytically almost as active as free pmAspAT. It could be reconstituted from isolated DnaK and pmAspAT. No complex was formed with mAspAT. Apparently, DnaK binds to the solvent-exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety. SO - Eur J Biochem 1992 Sep 15;208(3):699-704. Neg-159. PMID:2210055 | PIR:INHUR TI - Human insulin-receptor gene. Partial sequence and amplification of exons by polymerase chain reaction. AB - The partial sequence of the human insulin-receptor (hINSR) gene is presented. Using the gene sequence as a guide, we selected pairs of oligonucleotide primers from sites in the introns that flank each exon. These primers allowed each of the 22 exons of the hINSR gene to be amplified in vitro by the polymerase chain reaction. The sequences of the gene and oligonucleotide primers will facilitate studies of genetic variation in the hINSR gene and thereby increase our understanding of the role of this gene in the development of insulin-resistant states and glucose intolerance. SO - Diabetes 1990 Jan;39(1):123-8. Neg-160. PMID:2158100 | PIR:KIZMPO TI - Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize. AB - Analyses of genomic DNA and clones indicate that the pyruvate, orthophosphate dikinase (PPDK; ATP: pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1) gene family of maize (Zea mays L. subsp. mays, line B73) contains two members. Restriction site and DNA sequence comparisons between PPDK genomic and leaf cDNA clones have revealed which gene encodes the isozyme involved in C4 photosynthesis. The region flanking the 5' end of this gene contains two 30-base-pair (bp) repetitive elements that may be involved in its light-regulated expression. Sequence analysis of genomic and leaf cDNA clones has also shown that the entire 7.3-kDa PPDK chloroplast transit peptide is encoded in the 436-bp first exon. Northern blot experiments with probes specific for the first exon and the 3' end of the gene showed that the smaller PPDK transcripts in roots and etiolated leaves [3.0 kilobases (kb) vs. the 3.5-kb green leaf transcript] lack the sequence encoding the chloroplast transit peptide. In addition, results from cDNA library screens have confirmed that the root transcript is approximately 50-fold less abundant than the green leaf transcript. Finally, sequence comparisons among cDNA clones from green leaves and roots and genomic clones representing both members of the PPDK gene family demonstrate that the green leaf transcript encoding the C4 isozyme and the root transcript are derived from the same gene. SO - Proc Natl Acad Sci U S A 1990 Apr;87(8):3004-8. Neg-161. PMID:2157211 | PIR:TPHUN1 TI - Cloning of a cDNA for a major human protein-tyrosine-phosphatase. AB - We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) of human placenta. Degenerate oligonucleotides, based on the amino acid sequence of the protein, were used to amplify an internal fragment of the gene from human placental cDNA by the polymerase chain reaction. This fragment was then used to probe a human placental cDNA library. A 3.3-kilobase (kb) insert was isolated and sequenced. The insert has a single extended open reading frame that predicts a 435 amino acid protein of Mr approximately 50,000. From the amino terminus to residue 321, the deduced amino acid sequence is identical to that previously determined by peptide sequencing [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]; however, the sequence predicts that the protein contains an additional 114 amino acids not present in the reported peptide sequence. In vitro translation of the 3.3-kb insert produces a protein of Mr 56,000, in general agreement with the predicted size. The phosphatase gene appears to be present as a single copy in human genomic DNA and is transcribed into a 3.5-kb message in a variety of tissues. Cambridge, MA 02138. SO - Proc Natl Acad Sci U S A 1990 Apr;87(7):2735-9. Neg-162. PMID:1472036 | PIR:INHUR TI - Identification of a disulfide bridge connecting the alpha-subunits of the extracellular domain of the insulin receptor. AB - The alpha 2 beta 2 structure of the insulin receptor has previously been shown to involve one disulfide bridge between the alpha-subunits in the region containing Cys435, Cys468 and Cys524. We have digested the soluble extracellular domain of the insulin receptor with succinylated trypsin, partially separated the resulting peptides, and sequenced a number of fractions. The peptides containing Cys435 and Cys468 appeared in the same fraction, indicating that these two form a disulfide bond, and in another fraction we found the sequence of the peptide containing Cys524. Since it has been shown that the extracellular domain of the insulin receptor has no free thiols and since no other sequences containing cysteine were found in these fractions, we conclude that Cys524 forms a disulfide bond to the Cys524 in the other alpha-subunit. SO - Biochem Biophys Res Commun 1992 Dec 15;189(2):650-3. Neg-163. PMID:2455601 | PIR:A28822 TI - Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities. AB - Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154. SO - Cell 1988 Jul 15;54(2):171-7. Neg-164. PMID:2613245 | PIR:HSHUP1 TI - Chromosomal localization and structure of the human P1 protamine gene. AB - The human P1 protamine gene and mRNA were amplified with the use of the polymerase chain reaction and cloned into PTZ19R. The sequences were determined which revealed the presence of an intron. Southern and Northern hybridization analyses showed that the gene was single copy and that the mRNA was approximately 450 bases long. The gene was mapped to chromosome 16 with the use of a somatic cell hybrid panel and localized to the 21 region of the q arm by in situ hybridization of the human P1 protamine probe to human metaphase chromosomes. Detroit, Michigan 48202. SO - Genomics 1989 Oct;5(3):639-45. Neg-165. PMID:1840493 | PIR:S12318 TI - Cloning and characterization of the mitochondrial phosphate transport protein gene from the yeast Saccharomyces cerevisiae. AB - We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family. As PTP has a blocked N-terminus, we prepared three peptides. Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library. A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments. Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat. The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins. An N-terminal-extended signal sequence, as found in the beef protein, is absent. The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein. The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr. Mersalyl sensitivity has been retained and must be due to one of its three cysteines. Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein. Massachusetts 02114. SO - Biochemistry 1991 Jan 8;30(1):248-52. Neg-166. PMID:3194408 | PIR:MMHUE4 TI - Multiple protein 4.1 isoforms produced by alternative splicing in human erythroid cells. AB - Protein 4.1 is a multifunctional structural protein located in the erythrocyte membrane skeleton and in many nonerythroid cells. Molecular characterization of cloned protein 4.1 sequences from human reticulocytes has revealed the existence of multiple transcripts of the protein 4.1 gene that may encode a family of closely related protein isoforms. Several independently isolated cDNAs were sequenced and demonstrated to encode four different protein 4.1 species having identical primary sequences, except for the presence or absence of discrete peptides in the 8-kDa spectrin/actin binding domain (21 amino acids) and near the carboxyl terminus (43 and 34 amino acids). The same four protein 4.1 isoforms were detected when reticulocyte protein 4.1 mRNA sequences were reverse transcribed into cDNA and enzymatically amplified in vitro by using protein 4.1-specific oligonucleotide primers and the polymerase chain reaction. The finding of multiple protein 4.1 isoforms raises the possibility that the many binding functions ascribed to protein 4.1 may reside in distinct structural isoforms. Since only a single protein 4.1 gene appears to be expressed in erythrocytes, it is likely that these isoforms are produced by alternative mRNA splicing from a common protein 4.1 pre-mRNA. Multiple RNA splicing pathways are thus operative in the protein 4.1 gene even within a single cell lineage, human erythroid cells. 94143. SO - Proc Natl Acad Sci U S A 1988 Dec;85(23):9062-5. Neg-167. PMID:8604138 | PIR:I38344 TI - Genomic organization of M line titin and its tissue-specific expression in two distinct isoforms. AB - Titin is a 3000 kDa large protein of vertebrate striated muscle which extends from Z discs to M lines. Within the segment of titin that locates in the I band, tissue-specific isoforms are expressed by differential splicing in correlation to the sarcomeric ultrastructure. We have now searched the M-line region of titin for differential expression. The 20 kb section from the 3' end of the gene has been sequenced and contains 23 exons. Exon/intron organization is correlated to the modular organization of the titin protein. The six exons at the 3' end of the gene encode the M-line section of titin and are referred to as Mex1 to Mex6. Analysis of the RNAs expressed in different rabbit striated muscles reveals that the exon Mex5 is either included or excluded in the titin mRNA during splicing. The levels of inclusion of Mex5 vary between different types of striated muscles. Heart expresses (Mex5+)-titin, skeletal muscles co-express tissue-specifically distinct ratios of (Mex5+) and (Mex5-)-titins. In situ hybridization of whole-mount mouse embryos with Mex5 antisense RNA provide no evidence for the exclusion of Mex5 during embryonic development. We speculate that the establishment of differential splicing pathways of M-line titin late during development may correlate with and explain the postnatal development of different M-line fine structures in the different muscles. Comparison of titin gene sequences from different vertebrates reveals that the intron sequences located upstream of Mex3 and Mex5, referred to as Min-2 and Min-4, respectively, have remained strongly conserved during evolution. While the conservation of Min-4 may be explained by its participation in the regulation of the differential skipping of Mex5, the functional significance of the conservation of the Min-2 intron located upstream of Mex3 is yet unknown. SO - J Mol Biol 1996 Mar 1;256(3):556-63. Neg-168. PMID:6328443 | PIR:KABOSB KKBOB TI - Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs. AB - The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins. SO - Nucleic Acids Res 1984 May 11;12(9):3895-907. Neg-169. PMID:3576226 | PIR:KIRTC2 TI - Expression and properties of two types of protein kinase C: alternative splicing from a single gene. AB - Two complementary DNA's, encoding the complete sequences of 671 and 673 amino acids for subspecies of rat brain protein kinase C, were expressed in COS 7 cells. The complementary DNA sequence analysis predicted that the two enzymes are derived from different ways of splicing and differ from each other only in the short ranges of their carboxyl-terminal regions. Both enzymes showed typical characteristics of protein kinase C that responded to Ca2+, phospholipid, and diacylglycerol. The enzymes showed practically identical physical and kinetic properties and were indistinguishable from one of the several subspecies of protein kinase C that occurs in rat brain but not in untransfected COS 7 cells. Partial analysis of the genomic structure confirmed that these two subspecies of protein kinase C resulted indeed from alternative splicing of a single gene. SO - Science 1987 May 29;236(4805):1116-20. Neg-170. PMID:6687403 | PIR:ACRYD1 TI - Primary structures of beta- and delta-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences. AB - The nicotinic acetylcholine receptor (AChR) from fish electric organ and mammalian skeletal muscle is the best characterized neurotransmitter receptor (reviewed in refs 1-3). The AChR from the electroplax of the ray Torpedo californica consists of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 4-6); the apparent molecular weights of the alpha-, beta-, gamma- and delta-subunits are 40,000 (40K), 50K, 60K and 65K, respectively. Knowledge of the primary structures of these constituent polypeptides would facilitate the understanding of the molecular mechanism underlying the function of the neurotransmitter receptor. Recently, we have cloned cDNA for the alpha-subunit precursor of the T. californica AChR and have deduced the primary structure of this polypeptide from the nucleotide sequence of the cloned cDNA. Here we report the cloning and nucleotide analysis of cDNAs for the AChR beta- and delta-subunit precursors. The primary structures of the two polypeptides deduced from the cDNA sequences reveal conspicuous amino acid sequence homology among these and the alpha-subunits. The three subunits contain several highly conserved regions which may be essential for the receptor function or inter-subunit interaction. SO - Nature 1983 Jan 20;301(5897):251-5. Neg-171. PMID:1917919 | PIR:A40936 TI - Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia. AB - The oncoprotein 18 (Op18) gene encodes a proliferation-related cytosolic phosphoprotein, which is induced in normal lymphocytes following mitogenic stimulation. Studies of the Op18 gene are of particular interest because of the proposed role of Op18 protein in signal transduction and because of its occurrence in markedly increased amounts in acute leukemia cells. We have recently reported the cloning and sequencing of two cDNA clones for Op18 (1 and 1.5 kilobases). Both clones code for the same 149-amino acid polypeptide; however, they differ in their 3'-region as a result of alternative polyadenylation. We report here the sequencing of the Op18 gene and describe its expression in leukemia. The Op18 gene, which is 6.3 kilobases in length, is comprised of five exons and four introns and exhibits features that are common to other genes involved in cellular growth and proliferation. The increase in Op18 polypeptide in leukemia is associated with increased RNA transcription without gene amplification or rearrangement. Treatment of K562 leukemia cell line with hemin that induces terminal differentiation resulted in decreased expression of Op18. Our findings suggest that the high amount of Op18 protein in acute leukemia results from increased expression of a structurally unaltered gene. SO - J Biol Chem 1991 Sep 25;266(27):17747-53. Neg-172. PMID:3956509 | PIR:HSHUP2 TI - Human sperm protamines. Amino-acid sequences of two forms of protamine P2. AB - Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium X HCl containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gas-phase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine P1. The amino acid sequence of P2a is: (sequence; see text) SO - Eur J Biochem 1986 Apr 1;156(1):5-8. Neg-173. PMID:6299580 | PIR:TVCHS TVFV60 TI - Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. AB - We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA. SO - Cell 1983 Mar;32(3):881-90. Neg-174. PMID:1768865 | PIR:HSHUA1 TI - A novel divergently transcribed human histone H2A/H2B gene pair. AB - A genomic clone containing a novel closely linked human histone H2A/H2B gene pair has been isolated and sequenced along with extensive 5' and 3' flanking regions. Both genes are devoid of introns and code for core histone proteins. The nucleotide sequences are 84% and 87% homologous to the coding regions of a human genomic H2A and H2B gene, respectively. A comparison of the nucleotide-derived amino acid sequences shows that the histone H2A protein corresponds to the human H2A.1 subtype, whereas the H2B histone gene predicts an H2B protein sequence which is almost identical to the histone H2B.2 variant from human and bovine obtained by direct protein sequencing. The 3' flanking regions contain previously identified conserved sequence elements thought to be involved in transcription termination and processing of replication-dependent histone gene poly(A)- mRNAs. Primer extension analyses of the histone mRNAs encoded within this clone demonstrate that both genes are divergently transcribed from a 313 bp intergene promoter region. The spatial arrangement and orientation of two TATA-boxes, four CAAT-boxes, and one H2B-box within this region suggests that the linked genes share common promoter elements for transcriptional regulation. SO - DNA Seq 1991;1(6):409-13. Neg-175. PMID:6276759 | PIR:EQBOA TI - Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. AB - The nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla indicates that the precursor protein contains four copies of Met-enkephalin and one copy each of Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8, a previously undetected opioid peptide. The enkephalin and extended enkephalin sequences are each bounded by paired basic amino acid residues. Preproenkephalin may represent a multi-hormone precursor, like the corticotropin-beta-lipotropin precursor. SO - Nature 1982 Jan 21;295(5846):202-6. Neg-176. PMID:2877981 | PIR:FGHUA ITHUA2 TI - Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor. AB - A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI. SO - J Biol Chem 1986 Nov 25;261(33):15591-5. Neg-177. PMID:7569978 | PIR:I38344 TI - Titins: giant proteins in charge of muscle ultrastructure and elasticity. AB - In addition to thick and thin filaments, vertebrate striated muscle contains a third filament system formed by the giant protein titin. Single titin molecules extend from Z discs to M lines and are longer than 1 micrometer. The titin filament contributes to muscle assembly and resting tension, but more details are not known because of the large size of the protein. The complete complementary DNA sequence of human cardiac titin was determined. The 82-kilobase complementary DNA predicts a 3-megadalton protein composed of 244 copies of immunoglobulin and fibronectin type III (FN3) domains. The architecture of sequences in the A band region of titin suggests why thick filament structure is conserved among vertebrates. In the I band region, comparison of titin sequences from muscles of different passive tension identifies two elements that correlate with tissue stiffness. This suggests that titin may act as two springs in series. The differential expression of the springs provides a molecular explanation for the diversity of sarcomere length and resting tension in vertebrate striated muscles. SO - Science 1995 Oct 13;270(5234):293-6. Neg-178. PMID:2211593 | PIR:TIHUGK TI - Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions. AB - Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway. Albuquerque 87131. purification/pharmacology purification/pharmacology SO - J Biol Chem 1990 Oct 5;265(28):16786-93. Neg-179. PMID:2911561 | PIR:INHUR TI - Structure of the human insulin receptor gene and characterization of its promoter. AB - The human insulin receptor gene, INSR, and its promoter region have been isolated and characterized. The gene spans greater than 120 kilobase pairs (kbp) and has 22 exons. All introns interrupt protein coding regions of the gene. The 11 exons encoding the alpha subunit of the receptor are dispersed over greater than 90 kbp, whereas the 11 exons encoding the beta subunit are located together in a region of approximately 30 kbp. Three transcriptional initiation sites have been identified and are located 276, 282, and 283 bp upstream of the translation initiation site. In addition, a 247-bp fragment from the promoter region possessing 62.6% of the maximal promoter activity has been identified. This promoter-active fragment lacks a TATA-like sequence but has two possible binding regions for the transcriptional factor Sp1. Comparison of the exon structure of the tyrosine kinase domain of the INSR with the corresponding regions of the human SRC, ROS, and ERBB2 (NGL) protooncogenes indicates that the exon-intron organization of this region has not been well conserved. SO - Proc Natl Acad Sci U S A 1989 Jan;86(1):114-8. Neg-180. PMID:2384091 | PIR:HSHUP2 TI - Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3. AB - The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously. SO - Eur J Biochem 1990 Jul 31;191(2):449-51. Neg-181. PMID:3194415 | PIR:TVHUJN TI - Structure and chromosomal localization of the functional intronless human JUN protooncogene. AB - The JUN protooncogene encodes a protein that is functionally and biochemically identical to the transcription factor AP-1 (activator protein 1). To understand the structure and regulation of this important gene, a genomic clone of human JUN was isolated and its primary structure and transcription pattern were determined. Most surprisingly, the sequence of the genomic clone was found to be contiguous with the sequence of the JUN cDNA, suggesting that it lacks introns. RNase protection experiments confirm that JUN is an intronless gene that yields several transcripts due to 5' and 3' heterogeneities. Transfection experiments show that the cloned gene is functional, as it encodes a trans-acting factor that stimulates transcription of AP-1-dependent reporter gene. In situ hybridization was used to map JUN to chromosomal region 1p31-32. Interestingly, this region is frequently deleted in neuroblastomas, suggesting that elimination of AP-1 may play an important role in the pathogenesis of this disease. San Diego, La Jolla 92093. SO - Proc Natl Acad Sci U S A 1988 Dec;85(23):9148-52. Neg-182. PMID:4027356 | PIR:HSHUP1 TI - The amino acid sequence of human sperm protamine P1. AB - Human sperm protamines have been extracted from spermatozoa pooled from several donors, converted to their S-pyridylethylated derivatives and resolved into two major components, P1 and P2, by Bio-Rex 70 chromatography. Protamine P1 was further purified by Bio-Gel P-10 chromatography and sequenced directly on a gas phase protein sequencer for 43 residues. To complete the sequence, P1 was cleaved at methionine 36 and the C-terminal tetradecapeptide was purified by h.p.l.c. and sequenced completely. The 50 residue sequence is: (sequence see text) This sequence has a calculated molecular weight of 6674 and is homologous with four other published mammalian protamine sequences. SO - Biosci Rep 1985 May;5(5):383-91. Neg-183. PMID:1710598 | PIR:DVHUCF TI - Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31-q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns. Canada. SO - Genomics 1991 May;10(1):214-28. Neg-184. PMID:2498127 | PIR:QRBOT1 TI - Microtubule-associated protein tau. Identification of a novel peptide from bovine brain. AB - The microtubule-associated protein tau isolated from bovine brain was cleaved with CNBr and the 3 largest peptides of approx. 21, 19 and 18 kDa were obtained. Dephosphorylation of the CNBr digest of tau with alkaline phosphatase changed the electrophoretic mobility of these peptides to 19, 18 and 17 kDa. Amino acid sequencing of the total CNBr digest of tau revealed at least 3 sequences, two of which were highly homologous to previously published mouse and human tau sequences derived from cDNAs. A third amino acid sequence of 17 residues with heterogeneity at position 11 showed no homology with the cDNA-derived tau sequences. These studies suggest that the amino acid sequences of mammalian tau predicted from their cDNAs might be incomplete. Staten Island, NY 10314. purification SO - FEBS Lett 1989 May 8;248(1-2):87-91. Neg-185. PMID:624724 | PIR:TMRBA TI - The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. AB - The amino acid sequence of the large cyanogen bromide fragment (residues 11 to 127) derived from the NH2-terminal half of alpha-tropomyosin has been determined. This was achieved by automatic sequence analysis of the whole fragment as well as manual sequencing of fragments derived from tryptic digestion of the maleylated fragment and thermolytic, Myxobacter 495 alpha-lytic and Staphylococcus aureus protease digestion of the unmodified fragment. Methionine-containing overlap peptides have been isolated from tryptic digests of the maleylated protein as well as from S. aureus protease digests of the unmodified protein. Coupled with previously published information on the small cyanogen bromide fragments and methionine sequences of tropomyosin, these analyses have permitted the completion of the primary structure of the protein. The complete sequence differs by only 1 residue (Gln-24 instead of Glu-24) from that previously reported. Analysis of the sequence by several authors has permitted rational explanations for the stabilization of its coiled-coil structure, for the existence of its two chains in a nonstaggered arrangement, for a head-to-tail overlap of molecular ends of 8 to 9 residues, for the existence of 14 actin-binding sites on each tropomyosin molecule, and a suggestion for the site of binding of troponin-T. SO - J Biol Chem 1978 Feb 25;253(4):1137-48. Neg-186. PMID:2793849 | PIR:UDCH TI - Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution. AB - A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control. 08855. SO - J Biol Chem 1989 Oct 15;264(29):17164-9. Neg-187. PMID:1736899 | PIR:FGHUA TI - Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1-21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187. AB - The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood. SO - Biochem J 1992 Jan 15;281 ( Pt 2):519-24. Neg-188. PMID:3021205 | PIR:C1HURB TI - Nucleotide sequence of the cDNA coding for human complement C1r. AB - C1r is a zymogen of a serine protease that is involved in the activation of the first component of the classical pathway of the complement system. cDNAs coding for human C1r have been isolated from libraries prepared from poly(A) RNA from human liver and Hep G2 cells. From DNA sequence analysis, the overlapping cDNA inserts were shown to span 2493 nucleotides of the C1r mRNA, not including the poly(A) tail. The cDNA sequence coding for C1r contained a 5' noncoding region, 2115 nucleotides coding for a polypeptide precursor of 705 amino acids, and a 3' noncoding region. Some variability in the length of the 3' noncoding sequence was observed with the cDNA inserts, although most contained a polyadenylation signal followed by a poly(A) tail. The A or noncatalytic chain of C-1r, which originates from the amino-terminal end of the precursor molecule, contains a potential growth factor domain and two different pairs of internal repeats. One pair of these internal repeats is closely related to the amino-terminal sequence of C1s, while the other pair of repeats is homologous to the tandem repeats present in beta 2-glycoprotein I, complement factor B, the b subunit of factor XIII, and a single region present in the alpha 1 chain of haptoglobin. The B chain of C-1r contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases. SO - Biochemistry 1986 Aug 26;25(17):4855-63. Neg-189. PMID:3502720 | PIR:SBHUP TI - cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva. AB - On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used. SO - Am J Hum Genet 1987 Dec;41(6):1048-60. Neg-190. PMID:2325630 | PIR:A43803 TI - Coding sequence and flanking regions of the mouse vimentin gene. AB - Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a lambda gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5'- and 3'-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3'-untranslated sequences and the promoter region. Federal Republic of Germany. SO - Mol Gen Genet 1990 Mar;221(1):33-6. Neg-191. PMID:3814153 | PIR:KBBOA2 TI - Cloning and sequence analysis of bovine beta-casein cDNA. AB - A bovine beta-casein cDNA clone was isolated from a cDNA library prepared from mammary gland mRNA. Sequence analysis revealed 25 nucleotides (nt) of the 5' noncoding region, 672 nt of the complete sequence coding and a 3' region of approximately 500 nt. When the nucleotide sequence of bovine beta-casein cDNA is compared to rat beta-casein cDNA (5), a high degree of homology is observed in the first 100 nt corresponding to the signal peptide of the pre-beta-caseins. SO - Biochem Biophys Res Commun 1987 Jan 30;142(2):617-21. Neg-192. PMID:2452157 | PIR:TIHUGK TI - Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. AB - Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor Xa directly, and in a Xa-dependent fashion also inhibits the VIIa-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambda gt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind 125I-factor Xa. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambda P9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambda P9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The open reading frame encodes a signal peptide of 28 residues followed by a 32-kilodalton protein of 276 residues. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH2 terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH2 terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA (1.4 and 4.4 kb) which hybridize with LACI cDNA. SO - J Biol Chem 1988 May 5;263(13):6001-4. Neg-193. PMID:2927510 | PIR:TIHUGK TI - Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. AB - Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site. Medical Center, St Louis, Missouri 63110. SO - Nature 1989 Apr 6;338(6215):518-20. Neg-194. PMID:1704366 | PIR:SGHU1V TI - Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin. AB - The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule. Jolla, California 92037. SO - J Biol Chem 1991 Feb 15;266(5):2824-30. Neg-195. PMID:3619901 | PIR:A32794 TI - The human chromosomal protein HMG I contains two identical palindrome amino acid sequences. AB - The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA. SO - Biochem Biophys Res Commun 1987 Jul 31;146(2):725-30. Neg-196. PMID:2170848 | PIR:S12318 TI - Isolation and characterization of the gene for a yeast mitochondrial import receptor. AB - We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria. York 10021. SO - Nature 1990 Oct 4;347(6292):488-91. Neg-197. PMID:2081589 | PIR:HSHUP1 HSHUP2 TI - Genomic sequences of human protamines whose genes, PRM1 and PRM2, are clustered. AB - Protamines are small, arginine-rich proteins involved in the condensation of sperm chromatin. Using cDNA clones, we have isolated the genes for both human protamines, i.e., protamine 1 (PRM1) and protamine 2 (PRM2), from a human cosmid library. Each of these genes contains a single intron consisting of 91 and 163 bp, respectively. From the 5'-noncoding region of PRM1 664 bp and from the 5'-noncoding region of PRM2 902 bp were determined. Both genes contain typical TATAA and CAAT boxes at conventional distances from the transcription start points, which by using primer extension experiments could be assigned to nucleotides -91 and -110 for PRM1 and PRM2 genes, respectively. Comparison of the 5'-noncoding regions of PRM1 and PRM2 genes reveals 12 different motifs in common, 8 of which are clustered in both genes and could reflect regulatory elements for testis- and spermatid-specific gene expression. Both human genes have been found to be clustered at a distance of 4.8 kb. Comparison of the genomic organization of human and mouse protamine genes revealed greater similarities between the two in the 5'-noncoding region. Germany. SO - Genomics 1990 Sep;8(1):127-33. Neg-198. PMID:8250919 | PIR:A33430 TI - Common structural and expressional properties of vertebrate caldesmon genes. AB - We have determined the genomic structure of chicken caldesmon (CaD) gene. The gene, 100-150 kilobases long, is composed of 17 exons. Exons 1a-1, 1a-2, and 1a-3 encode the 5'-terminal sequence specific to the mRNAs for CaDs expressed in gizzard. Exon 1b encodes the 5'-terminal sequence of the brain l-CaD and locates downstream of exons 1a-1, 1a-2, and 1a-3. The genomic construction of the chicken CaD resembles with that of the human CaD. Exon 3 of chicken CaD gene possesses the unique structure similar to that of human CaD gene; the common domain in both h- and l-CaDs (amino acid residues 74-199 for h-CaD and residues 66-191 for l-CaD) and the central repeating domain specific to h-CaD (amino acid residues 200-419) are encoded in exons 3a and 3b, respectively. Of particular interest is that the two consensus 5'-splice sites are found in the borders between exons 3a and 3b, and exon 3b and intron. Therefore, the expressional regulation between h- and l-CaDs can be explained by selection of these 5'-splice sites. Alternative 3'-splice sites also exist at intron/exon junction of exon 14 and the difference in selection of the sites would induce the specific Ala-508 insertion in the brain l-CaD. Medical School, Japan. SO - Biochem Biophys Res Commun 1993 Nov 30;197(1):145-53. Neg-199. PMID:2469626 | PIR:HHHU86 HHMS86 TI - Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumour-specific transplantation antigens. AB - Mouse cDNA clones have been isolated with the help of Drosophila melanogaster 82-kDa heat-shock protein (Hsp82)-coding sequences as hybridization probe. Sequencing of the overlapping mouse clones reveals a long open reading frame (ORF) that encodes a polypeptide of 83.3 kDa which shows about 80% similarity to the respective Drosophila Hsp82 amino acid sequence. The N-terminal half of this cDNA cross-hybridizes to a different class of mouse cDNA clones indicating a related gene. Northern blot hybridization experiments reveal a 2.6-kb poly(A)+RNA when probed with the hsp84 clone and a 2.85-kb signal with the hsp84-related cDNA. The amino acid sequences deduced from the contiguous ORF of the hsp84 and the hsp84-related cDNA coincide with the N-terminal sequence of formerly identified 84-kDa and 86-kDa tumour-specific transplantation antigens (Ullrich et al., 1986). In addition, the amino acid composition of the putative 84-kDa mouse Hsp described here is very similar to that of the 84-kDa tumour antigen described by Ullrich et al. (1986). Both observations corroborate the assumption that these Hsps are identical to the described 84-kDa and 86-kDa tumour-specific transplantation antigens. Using these mouse hsp gene clones as hybridization probes we also isolated the corresponding pair of human cDNA clones. Comparison of the respective sequences reveals a strong evolutionary constraint on these two genes in mouse and man. SO - Gene 1988 Dec 30;74(2):491-501. Neg-200. PMID:1939157 | PIR:GEBOM GEHUM TI - Carboxyl-terminal proteolytic processing of matrix Gla protein. AB - The present study was undertaken to determine the extent of COOH-terminal proteolytic processing in matrix Gla protein (MGP), a 10-kDa protein which contains 5 residues of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla). Two forms of MGP were isolated from demineralization and urea extracts of bovine cortical bone, one 79 residues in length with the COOH terminus Phe-Arg-Gln and the other 83 residues in length with the COOH terminus Phe-Arg-Gln-Arg-Arg-Gly-Ala. The 84-residue form of bovine MGP predicted from the message structure could not be detected in the bone extracellular matrix extracts, and it therefore seems probable that the lysine at position 84 was removed by the action of a carboxypeptidase B-like enzyme prior to secretion. A plausible sequence of proteolytic cleavages that could generate the 79-residue form of MGP would be a trypsin-like cleavage at Arg80-Arg81 or Arg81-Gly82 followed by carboxypeptidase B-like cleavage to remove COOH-terminal arginine(s). Since essentially equal amounts of the 79- and 83-residue forms of MGP were also detected in bovine articular cartilage and plasma, it seems likely that the COOH-terminal processing events identified in bone apply to many of the other tissues which synthesize this protein. Only one form of MGP was detected in human bone extracts, a 77-residue protein that lacks the COOH-terminal residues Arg-Lys-Arg-Arg-Gly-Thr-Lys. This shortened version of human MGP is consistent with the proposed model for COOH-terminal processing, since the amino acid substitution in the COOH terminus of the human protein, Lys79 for Gln79, would allow removal of the additional basic residues from the human MGP COOH terminus by the action of the carboxypeptidase B-like enzymic activity. Recent studies have shown that MGP is strongly induced by retinoic acid in fibroblasts, chondrocytes, and osteoblasts, a response which suggests that MGP mediates an action of retinoic acid on an aspect of cell growth or differentiation. If this hypothesis is true, the present evidence for complex COOH-terminal processing events could provide a means to regulate the as yet unknown activity of MGP in the extracellular environment in a mechanism similar to the activation of hormones such as anaphlotoxins and kinins. 92093-0322. SO - J Biol Chem 1991 Nov 5;266(31):21145-9. Neg-201. PMID:1889406 | PIR:HSHUP2 TI - Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei. AB - Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed. Creteil, France. SO - Eur J Biochem 1991 Sep 1;200(2):387-92. Neg-202. PMID:1658736 | PIR:KABOSB TI - Genomic organization of the bovine alpha-S1 casein gene. AB - We report the sequence of the complete bovine alpha-s1 casein gene eludicating for the first time the genomic organization of an alpha-s type casein gene. Extending over 17508 bp the gene is split into 19 exons, ranging in size from 24 bp to 385 bp. Except for the translational stop codon not a single coding triplet of the alpha-s1 reading frame is disrupted by any of the splice junctions, which all confirm to known splice consensus sequences. Nine out of 16 coding exons begin with a 'GAX' codon, specific for glutamate. Splicing of this codon from exon 10 to the preceding exon creates a major phosphorylation site. An intron-exon-intron stretch of 154 bp comprising exons 10 and 13 is found precisely duplicated. Associated with the gene, copies of 8 atriodactyla retroposons are found, 6 of which are interspersed into the sequences of the three longest introns. We discuss the possibility that three functional parts of the gene have been recruited and evolutionary conserved at a time before gene diversification gave rise to the separate evolution of alpha- and beta-type casein-genes. Giessen, FRG. SO - Nucleic Acids Res 1991 Oct 25;19(20):5591-6. Neg-203. PMID:6773933 | PIR:PEMQAJ TI - Monkey pepsinogens and pepsins. IV. The amino acid sequence of the activation peptide segment of Japanese monkey pepsinogen. AB - The complete amino acid sequence of the activation peptide segment of the major component of Japanese monkey pepsinogens was determined. The pepsinogen was converted to pepsin by incubation at pH 2.0 and 14 degrees C for 17 min. The activation peptides were separated from the resulting pepsin and purified by chromatography on columns of sulfopropyl (SP)-Sephadex. One 25-residue peptide and two 22-residue peptides were isolated and their amino acid sequences were determined. The results showed that the former peptide was derived from the amino-terminal half (residues no. 1 to 25) and the latter peptides from the carboxyl-terminal half (residues no. 26 to 47) of the activation segment. The latter two peptides were identical except for a single amino acid replacement. The complete amino acid sequence of the whole activation peptide segment was thus deduced as follows: Ile1-Ile-Tyr-Lys-Val-Pro-Leu-Val-Arg-Lys10-Lys-Ser-Leu-Arg-Arg-Asn-Leu-Ser -Glu- His20-Gly-Leu-Leu-Lys-Asp-Phe-Leu-Lys-Lys-His30-Asn-Leu-Asn-Pro-Ala-Ser-Ly s-Tyr -Phe-Pro40-LysGln-Ala-Glu-Ala-Pro-Thr-Leu47. The whole chain was composed of 47 residues, and cleaved into two polypeptides by the cleavage of Asp25-Phe26 bond during activation. Two amino acids were detected at residue-41, i.e., glutamine and lysine, indicating the presence of two closely related pepsinogens in the sample used. The total number of residues (47) of the whole activation peptide segment is 3 and 2 residues larger than those of porcine pepsinogen and bovine pepsinogen, respectively. The differences are 15 and 22 residues as compared with porcine pepsinogen and bovine pepsignogen, respectively. SO - J Biochem (Tokyo) 1980 Jul;88(1):9-16. Neg-204. PMID:2719677 | PIR:A32541 B32541 TI - Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2). AB - We screened a human parotid gland cDNA library with mixed synthetic oligonucleotide probes representing a central coding region common to histatins 1 and 3. Sequence analysis of 12 histatin cDNA clones strongly suggests that the histatin protein family is encoded by at least two closely related loci (HIS1 and HIS2) such that histatins 1 and 3 are primary products of HIS1(1) and HIS2(1) alleles, respectively, and that histatins 4-6 are derived from histatin 3 by proteolysis. We present additional data indicating that histatin 2 may represent the non-phosphorylated form of histatin 1. SO - Biochem Biophys Res Commun 1989 Apr 28;160(2):495-502. Neg-205. PMID:6945574 | PIR:S05207 TI - Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: desmin and vimentin. AB - Although all intermediate-size filaments (10-nm filaments) seem to show similar morphology and share a number of biochemical properties, different cell- and tissue-specific subclasses have been distinguished by immunological experiments and by differences in apparent molecular weights and isoelectric points of the major constituent proteins. In order to understand the degree of possible homology between these proteins, we have begun amino acid sequence analysis of the polypeptides. Here we characterize a large fragment of chicken gizzard and pig stomach desmin as well as the corresponding fragment from porcine eye lens vimentin. The fragments are situated at the carboxyl end and consist of 138-140 amino acid residues--i.e., some 28% of the corresponding polypeptide chains. The results show that the two immunologically distinct porcine proteins are different gene products. They show a related amino acid sequence but differ in 36% of the residues present in the carboxy-terminal region. Thus tissue specificity overrides species divergence. These results are discussed in the light of previous immunological experiments. They lend further support to the hypothesis that intermediate filaments belong to a multigene family, which is expressed in line with certain rules of differentiation during embryogenesis. SO - Proc Natl Acad Sci U S A 1981 Jul;78(7):4120-3. Neg-206. PMID:8918249 | PIR:F70584 TI - A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system. AB - We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease. SO - Gene 1996 Oct 17;176(1-2):171-6. Neg-207. PMID:7882970 | PIR:S54304 TI - Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium. AB - Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals. They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain. Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea. The protein CheAH.s. (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g. 34% identity with CheA from Bacillus subtilis). Antibodies were raised against CheAH.s. as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s. in H. salinarium. As has been observed for other halophilic proteins, CheAH.s. has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa. Deletion of a part of the cheAH.s. gene leads to loss of both chemotactic and phototactic responses in H. salinarium as measured by swarm plate assays, motion analysis and tethering experiments. This indicates that CheAH.s. plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors. SO - EMBO J 1995 Feb 15;14(4):667-73. Neg-208. PMID:6662498 | PIR:UDCH UDHU TI - Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white. AB - Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme. SO - Hoppe Seylers Z Physiol Chem 1983 Nov;364(11):1487-96. Neg-209. PMID:661981 | PIR:OACH TI - Sequence of chicken ovalbumin mRNA. AB - The complete sequence of chicken ovalbumin mRNA is presented; it is 1,859 residues long, excluding its terminal 'cap' and poly(A). The region coding for ovalbumin lies close to the 'cap' but is separated from the poly(A) by an extensive 3' noncoding region of 637 nucleotides which may have no function that is precisely dependent on its sequence. SO - Nature 1978 Jun 29;273(5665):723-8. Neg-210. PMID:3232124 | PIR:FGHUA TI - A direct-acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix: effects on various components of the human blood coagulation and fibrinolysis systems. AB - A direct acting fibrinolytic enzyme (fibrolase) has been isolated from venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Time-course experiments established that the venom enzyme cleaves primarily the A alpha-chain of human fibrinogen and fibrin between the Lys-413 and Leu-414 position. The B beta-chain is cleaved more slowly, while the gamma-chain is minimally affected. The cleavage pattern of fibrinogen and fibrin clearly varies from plasmin cleavage of the same molecules. The enzyme does not activate plasminogen or protein c and it is thus different from "Protac", a protein c activator isolated from the same venom. Biochemistry, Los Angeles. SO - Thromb Res 1988 Dec 15;52(6):541-52. Neg-211. PMID:2605252 | PIR:ACRYD1 TI - Proteolytic fragments of the nicotinic acetylcholine receptor identified by mass spectrometry: implications for receptor topography. AB - A triple-state quadrupole or a tandem quadrupole Fourier-transform mass spectrometer was used to detect and sequence the peptides released by proteolytic cleavage of the acetylcholine receptor (AcChR) from Torpedo californica electroplax. Fragments in mass range up to 3479 daltons were characterized on the above instrumentation and used to determine proteolytically accessible sites on the receptor. These data were consistent with the cleavage points determined for membrane-bound fragments of the same AcChR samples using gas-phase microsequencing. Each subunit of the receptor is readily cleaved near the C-terminus in the region between the proposed transmembrane hydrophobic alpha-helices MIII and MIV. This region includes the putative regulatory phosphorylation sites and the amphipathic alpha-helix. Cleavage is also observed in the N-terminal domain, but occurs much more slowly than in the C-terminal region. No cleavage was detected in the middle third of the receptor, which includes the proposed transmembrane alpha-helices MI and MII. An evaluation of these data in terms of the transmembrane topography of the AcChR peptides is consistent with a synaptic or extracellular disposition for the region between MIII and MIV. SO - Biochemistry 1989 Nov 14;28(23):9184-91. Neg-212. PMID:3317405 | PIR:GERTM1 TI - Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase. AB - Matrix Gla protein (MGP), a low molecular weight protein found in bone, dentin, and cartilage, contains 5 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid (Gla). We have used antibodies raised against MGP and oligonucleotide probes to screen a lambda gt11 cDNA library constructed from the rat osteosarcoma cells (line ROS 17/2) that had been pretreated with 1 alpha,25-dihydroxyvitamin D3. By sequencing several cloned cDNAs, we established a 523-base-pair sequence that predicts an 84-residue mature MGP and a 19-residue hydrophobic signal peptide. The 84-residue mature rat MGP predicted from the cDNA sequence has an additional 5 residues at its C terminus (-Arg-Arg-Gly-Ala-Lys) not seen in the sequence of MGP isolated from bovine bone. The structure of rat MGP provides insight into the mechanisms by which the vitamin K-dependent gamma-carboxylase recognizes substrate. The present studies show that MGP, unlike other vitamin K-dependent proteins, lacks a propeptide. The absence of an MGP propeptide demonstrates that gamma-carboxylation and secretion of vitamin K-dependent proteins need not be linked to the presence of a propeptide or to its proteolytic removal. The propeptides of other vitamin K-dependent proteins are structurally homologous, and there is evidence that this homologous propeptide domain is important to substrate recognition by the gamma-carboxylase. Mature MGP has a sequence segment (residues 15-30) that is homologous to the propeptide of other vitamin K-dependent proteins and probably serves the same role in gamma-carboxylase recognition. Rat MGP also has a second sequence that has recently been identified in all known vitamin K-dependent vertebrate proteins, the invariant unit Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys (EXXXEXC). Since the glutamic residues in this unit are sites of gamma-carboxylation, it has been suggested that the EXXXEXC unit could allow the gamma-carboxylase to discriminate between substrate and product. The demonstration that two structures common to vitamin K-dependent proteins, the homologous propeptides domain and the invariant EXXXEXC unit, are in mature MGP indicates that des-gamma-carboxy-MGP should be an excellent in vitro gamma-carboxylase substrate for analysis of mechanisms involved in substrate recognition and product dissociation. 92093. SO - Proc Natl Acad Sci U S A 1987 Dec;84(23):8335-9. Neg-213. PMID:2552037 | PIR:A34957 TI - ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Molecular cloning and nucleotide sequence. AB - A cDNA clone for the mRNA of bovine ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS-PAGE) was isolated from a modified Okayama-Berg plasmid library. Transformed Escherichia coli colonies were screened by in situ colony hybridization with 2 different oligonucleotide probes derived from the amino acid sequence of the bovine protein. Sequence analysis of the longest cDNA clone, pTKAI [2407 nucleotides plus a poly(A) tail], revealed a 267-nucleotide-long coding region in agreement with the bovine ARPP-21 amino acid sequence (Williams et al., 1989). Southern blot analysis of total bovine genomic DNA raised the possibility that there may be 2 genes coding for ARPP-21. Northern blot analysis of total cellular RNA from bovine caudate nucleus and other brain regions demonstrated the existence of 2 major mRNA species, 2.5 and 1.0 kb in length, probably derived from use of alternate polyadenylation sites. There was a differential expression of these 2 mRNAs within the brain. Both ARPP-21 mRNAs were most abundant in the caudate nucleus, where the concentration of the protein is highly enriched. New York, New York 10021. SO - J Neurosci 1989 Oct;9(10):3638-44. Neg-214. PMID:6090423 | PIR:QRECCY TI - Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein. AB - We overexpressed the CheY protein by fusing the cheY gene to the tryptophan promoter from Serratia marcescens. Expression of the trp promoter-cheY fusion and subsequent purification of the protein resulted in the isolation of up to 20 mg of homogeneously pure CheY protein from 100 mg of the cytoplasmic supernatant fraction. Purification of the CheY protein was accomplished by exploiting the affinity of CheY protein to cibacron blue dye and molecular sieve chromatography. Preliminary biochemical characterization of the pure CheY protein revealed specific interactions with S-adenosylmethionine and cibacron blue dye. Additional kinetic analysis showed that CheY protein inhibits EcoRI methyltransferase. The amino acid composition of the CheY protein predicted by the DNA sequence of the cheY gene and the amino acid analysis of the CheY protein were in agreement, confirming the authenticity of the purified CheY protein. SO - J Bacteriol 1984 Oct;160(1):36-41. Neg-215. PMID:2553722 | PIR:TIHUGK TI - Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma. AB - The lipoprotein-associated coagulation inhibitor (LACI) has been isolated from human plasma using a combination of hydrophobic, ion-exchange, and affinity chromatography. The final purification required was greater than 500,000-fold with a yield of 13%. Plasma LACI, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains major bands at 40 and 46 kDa and minor bands at 55, 65, 75, 90, and approximately 130 kDa. All of the molecular weight forms are recognized by antibodies to LACI's amino and carboxyl termini and are able to inhibit the factor VII(a)-tissue factor complex and factor Xa. Plasma LACI, reduced with beta-mercaptoethanol, migrates on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis as a doublet at 42 kDa and has an amino-terminal sequence essentially identical to that of HepG2 LACI. The difference in size between reduced plasma LACI (42 kDa) and HepG2 LACI (47 kDa) may be related to differing degrees of N-linked glycosylation. The 46-kDa and larger forms of unreduced plasma LACI are associated with apolipoprotein A-II (apoA-II) in mixed disulfide linkages. Studies using isolated lipoproteins show that low density lipoprotein (LDL) contains primarily the 40-kDa form of LACI, whereas high density lipoprotein (HDL) contains primarily the 46-kDa form of LACI (LACI/apoA-II complexes). Gel filtration of a fresh plasma sample showed approximately 50% of plasma LACI to be associated with LDL/very low density lipoprotein, 44% with HDL, and the remaining 6% to not be associated with lipoproteins. Jewish Hospital, St. Louis, Missouri 63110. purification/pharmacology SO - J Biol Chem 1989 Nov 5;264(31):18832-7. Neg-216. PMID:3763441 | PIR:GMDG JS0426 TI - Sequences of gastrins purified from a single antrum of dog and of goat. AB - Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only approximately 5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: Dog less than ELGLQGPPQLVADLSKKQGPWMEEEEAAYGWMDF# Goat less than ELGLQDPPHMVADLSKKQGPWVEEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog. SO - Peptides 1986 Jul-Aug;7(4):689-93. Neg-217. PMID:8840504 | PIR:S12318 TI - Analysis of a 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI. AB - We have sequenced a 61.989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase. Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI. SO - Yeast 1996 Jul;12(9):869-75. Neg-218. PMID:3044927 | PIR:PEPG TI - Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein. AB - A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule. University, Japan. SO - Gene 1988 May 30;65(2):285-92. Neg-219. PMID:3447155 | PIR:INHUR TI - Partial amino acid sequence analyses of human placental insulin receptor. AB - Tryptic peptides were isolated from human insulin receptor (1.8 nmol) by sequential reverse-phase HPLC on SynChropak RP-C8 and Vydac C-18. Nearly 20 peptides were analyzed for amino acid composition, which revealed that the yield of tryptic peptides averaged 300 pmol. By gas-phase microsequencing five peptides were sequenced. N-acetylglucosamine was found in two peptides. These data are compared with the amino acid sequence of the insulin receptor deduced from cloned cDNA encoding for human insulin receptor. of Hope, Duarte, CA 91010-0269. SO - Protein Seq Data Anal 1987;1(1):3-6. Neg-220. PMID:2545626 | PIR:F70584 TI - Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis. AB - Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule. None of the antibodies bound to the N-terminal 117-amino-acid peptide. SO - Infect Immun 1989 Aug;57(8):2481-8. Neg-221. PMID:3466902 | PIR:A27335 TI - Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis. AB - Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls. SO - J Clin Invest 1987 Jan;79(1):275-81. Neg-222. PMID:6173760 | PIR:EQBOA TI - Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides. AB - Molecular cloning and DNA sequencing have yielded considerable structural information about proenkephalin. All previously characterized intermediate peptides of the enkephalin pathways in bovine adrenal medulla have now been aligned into an unambiguous primary structure. Two basic amino acid residues serve as processing signals for release of each of the different components. SO - Nature 1982 Jan 21;295(5846):206-8. Neg-223. PMID:2428667 | PIR:KIRTC1 KIRTC2 TI - Two types of complementary DNAs of rat brain protein kinase C. Heterogeneity determined by alternative splicing. AB - Two types of complementary DNA clones for rat brain protein kinase C were isolated. These clones encode 671 and 673 amino acid sequences, which differ from each other only in the carboxyl-terminal regions of approx. 50 amino acid residues. This difference seems to result from alternative splicing. Elucidation of the sequences of these cDNA clones as well as some peptides from the purified rat brain enzyme suggests the existence of an additional species of protein kinase C in this tissue. It is attractive to imagine that the heterogeneity of protein kinase C may reflect diverse pathways of signal transduction into the cell. SO - FEBS Lett 1986 Oct 6;206(2):347-52. Neg-224. PMID:1457396 | PIR:D44234 FGHUA TI - Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits. AB - Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology. 10021. SO - Biochemistry 1992 Dec 8;31(48):11968-72. Neg-225. PMID:6368553 | PIR:FMSP32 TI - Molecular architecture of the rapidly metabolized 32-kilodalton protein of photosystem II. Indications for COOH-terminal processing of a chloroplast membrane polypeptide. AB - The molecular architecture of the rapidly metabolized 32-kilodalton chloroplast protein was investigated. Modified proteolytic techniques were applied to construct a cleavage map for this photosynthetic membrane component. The portion which anchors the protein to the membrane was shown to be rich in hydrophobic amino acid residues, while the surface-exposed portion was richer in polar residues. The amino to carboxy polarity of the map was established by comparing oligopeptide radiolabeling data with the polypeptide sequence decoded from the gene. This showed that the anchor domain contains the NH2 terminus, and the exposed domain the COOH terminus. The 32-kilodalton protein was previously demonstrated to derive from a 33.5-kilodalton precursor. We now show that the precursor molecule has a COOH-terminal oligopeptide extension. A model relating precursor cleavage to membrane integration is presented. COOH-terminal processing is proposed to ensure a correct sequence of events for assembly of the 32-kilodalton protein into the photosynthetic membrane. SO - J Biol Chem 1984 Mar 25;259(6):3900-8. Neg-226. PMID:6286662 | PIR:OKBO2C TI - Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates. AB - The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198. SO - J Biol Chem 1982 Sep 25;257(18):10575-81. Neg-227. PMID:2002011 | PIR:QRECCS TI - Tandem translation starts in the cheA locus of Escherichia coli. AB - The cheA locus of Escherichia coli encodes two protein products, CheAL and CheAS. The nucleotide sequences of the wild-type cheA locus and of two nonsense alleles confirmed that both proteins are translated in the same reading frame from different start points. These start sites were located on the coding sequence by direct determination of the amino-terminal sequences of the two CheA proteins. Both starts are flanked by inverted repeats that may play a role in regulating the relative expression rates of the CheA proteins through alternative mRNA secondary structures. SO - J Bacteriol 1991 Mar;173(6):2116-9. Neg-228. PMID:862621 | PIR:WPSAHP TI - The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 1. Amino-acid sequence of the phosphocarrier protein HPr. AB - The primary structure of the histidine-containing phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system from Staphylococcus aureus was determined by automated Edman degradation. The complete sequence was deduced from the direct analysis of the protein by automated Edman degradation in a liquid-phase sequencer of Edman and from the sequence of tryptic, thermolytic and cyanogen bromide peptides as obtained by automated Edman degradation in a solid-phase sequencer of Laursen. The amino-acid sequence was found to be Met-Glu-Gln-Asn-Ser-Tyr-Val-Ile-Ile-Asp-Glu-Thr-Gly-Ile-His-Ala-Arg-Pro-Al a-Thr-Met-Leu-Val-Gln-Thr-Ala-Ser-Lys-Phe-Asp-Ser-Ile-Asp-Gln-Gly-Gly-Tyr- Asp-Ser-Met-Gln-Leu-Lys-Ser-Leu-Gly-Val-Gly-Lys-Asp-Glu-Glu-Ile-Thr-Ile-Tm -Ser-Ala-Asp-Lys-Lys-Glu-Gly-Leu-Thr-Lys-Met-Ser-Ile-Val. The 70 residues correspond to a molecular weight of 7685. The one histidine involved in the phosphotransfer reaction of this protein was found at position 15 as part of a region of the sequence which has no predictable secondary structure. It is suggested that this protein belongs to the group of male proteins with the active center located on a protrusion rather than a cleft. SO - Eur J Biochem 1977 May 2;75(1):275-86. Neg-229. PMID:8076687 | PIR:F2SPD2 FMSP32 TI - Nearest neighbor analysis of D1 and D2 subunits in the photosystem II reaction center using a bifunctional cross-linker, hexamethylene diisocyanate. AB - A cross-linked product between the D1 and D2 subunits was generated by treating isolated spinach PS II reaction center with a bifunctional cross-linker, 1,6-hexamethylene diisocyanate. To obtain information on the contact site(s) between D1 and D2 proteins, the cross-linked product was cleaved by CNBr, the resultant fragments were separated using a reverse-phase HPLC, and then the partial sequence of the cross-linked polypeptide fragments were determined. By comparing the results with the deduced amino acid sequence of the D1 and D2 proteins from spinach, it is concluded that the C-terminal domains of the D1 subunit (D308-A334) and that of the D2 subunit (Y297-L353) are in close proximity. SO - FEBS Lett 1994 Aug 29;351(1):27-30. Neg-230. PMID:8359684 | PIR:UDCH TI - Isolation and characterization of the chicken cystatin-encoding gene: mapping transcription start point and polyadenylation sites. AB - A 6.9-kb fragment containing coding sequences for chicken egg white cystatin (CsnEW) was isolated from a chicken genomic library using the CsnEW cDNA as a probe. The gene is approximately 2.4 kb in length; it contains three exons, two introns and two polyadenylation signals. The exon-intron arrangement corresponds exactly with those of other members of the Csn superfamily. The sequence of the 5' flanking region contains two SP1-binding sites and a high G+C content suggestive of a housekeeping gene. All tissues studied express CsnEW mRNA; Northern analysis showed that CsnEW mRNA levels are most abundant in lung and least abundant in liver and spleen. Mapping of the 3' end of the CsnEW mRNA isolated from brain tissue resolved two CsnEW mRNA species. The larger transcript resulting from the use of the second polyadenylation signal was more abundant than the smaller transcript. Determination of the transcription start point (tsp) of CsnEW mRNA by primer extension and RNase protection assays showed that CsnEW mRNA from a number of chicken tissues was approximately 40-50 nucleotides shorter than that predicted from the CsnEW cDNA isolated from chicken oviduct. Louisville School of Medicine, KY 40292. SO - Gene 1993 Aug 25;130(2):175-81. Neg-231. PMID:6194898 | PIR:VEHY TI - The structure of the vimentin gene. AB - The structure of the chromosomal gene encoding the intermediate filament protein vimentin is described. This gene, which is present as a single copy in the hamster genome, comprises about 10 kb of DNA and contains more than 80% of intron sequences. S1 mapping and sequence analysis reveal nine exons with a total length of 1848 nucleotides. For the complete primary structure of hamster vimentin, 464 amino acids are predicted, giving a molecular weight of 53,500 daltons. The intron positions are at codons 186, 206/207, 238/239, 292/293, 334/335, 408, 423, and 451/452. The overall homology with chicken desmin is 60% and is even higher in the central (alpha-helical) regions of both molecules. Cross-hybridization at the DNA level, however, is low. Comparison of the amino acid sequence of vimentin with prekeratin sequences shows that there is lesser homology of primary structure, but both the position and size of alpha-helical regions are strongly conserved. At the 5' end of the gene there is a consensus promoter sequence. The first AUG start codon is found 132 nucleotides downstream of the estimated cap site. The 3' nontranslated sequence shows homologies with the chicken vimentin gene. An interesting feature of the vimentin gene is a stretch of 44 nucleotides of alternating dC and dA within intron 2 that may form left-handed Z-DNA. SO - Cell 1983 Nov;35(1):215-23. Neg-232. PMID:3003084 | PIR:IQECDK TI - Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product. AB - The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975. SO - J Biol Chem 1986 Feb 5;261(4):1778-81. Neg-233. PMID:6148101 | PIR:KBBOA2 TI - Neoglycoproteins: in vitro introduction of glycosyl units at glutamines in beta-casein using transglutaminase. AB - Exploring different methods for preparing neoglycoproteins with a specific number of oligosaccharides in specific positions, we have used guinea pig liver transglutaminase to incorporate glycosyl units into glutamine residues in beta-casein. In order to prevent epsilon-(gamma-glutamyl)lysine cross-link formation, the lysine residues of beta-casein were first blocked either by amidination with ethyl acetimidate or by acylation with succinic anhydride. The glycosyl donor substrates prepared for this work were maltotriose reductively aminated with cadaverine, N-(Glc-Glc-glucitol-1)-cadaverine, and an asparaginyl nonasaccharide from ovalbumin modified with a 6-aminohexanoyl group at the alpha-amino group. The transglutaminase-catalyzed incorporation of these two donors into the beta-casein derivatives was monitored in comparison to the incorporation of the commonly used transglutaminase substrate dansylcadaverine under conditions of optimal incorporation (multiple additions of enzyme, large excess of donor, and long incubation time). For both dansylcadaverine and Glc-Glc-Glc(OH)-cadaverine, 5 and 8 mol of donor were incorporated per mol of amidinated and succinylated beta-casein, respectively. Competition experiments showed that the two donor substrates are incorporated into the same glutamine sites. Partial sequencing of the glycosylated beta-casein permitted the identification of glutamine residues 56, 79, 167, 175, and 194 as the primary sites of incorporation in amidinated casein with residues 54 and 182 as possible sites for partial glycosylation. The results are consistent with a specific glycosylation of only selected glutamines in this transglutaminase-catalyzed process.(ABSTRACT TRUNCATED AT 250 WORDS) SO - Biochemistry 1984 Jul 31;23(16):3759-65. Neg-234. PMID:3004934 | PIR:SGHU1V TI - Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. AB - cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways. SO - EMBO J 1985 Dec 1;4(12):3153-7. Neg-235. PMID:9634230 | PIR:BVMYBA F70584 G70887 H70887 S15205 TI - Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. AB - Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the blased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation. stcole@pasteur.fr SO - Nature 1998 Jun 11;393(6685):537-44. Neg-236. PMID:2162844 | PIR:A56968 TI - Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. AB - The cloning and characterization of cDNAs for the catalytic subunit of calcineurin (CN) from murine and human brain libraries were carried out using nonisotopic methods. A murine cDNA clone encoding a protein of 521 amino acids (Mr approximately 58,650) was isolated; overlapping clones established a 3'-untranslated region of 554 base pairs preceding the poly(A) tail. Homologous cDNAs from human brain showed greater than 92% nucleotide sequence identity in both coding and non-coding regions with greater than 99% conservation of amino acid sequence. A second class of cDNAs lacking a specific 30-base pair region following the calmodulin-binding domain was found in four murine and human libraries. Oligonucleotide probes for both cDNA isoforms hybridized to mRNA from several brain regions indicating the existence of transcripts in vivo. The nucleotide sequences of the two forms were identical except for the inserted sequence, and Southern blot analysis of mouse and rat DNA was consistent with their having originated from the same gene; these data suggest that alternative splicing may give rise to molecular isoforms of the catalytic subunit in brain. Northern blots showed a predominant mRNA for CN in most tissues of approximately 4.0 kilobases (kb) with lower amounts of a 3.6-kb species. Brain showed 10 times more of these mRNAs than skeletal muscle while other tissues had less than or equal to 5% that in brain. In testis, multiple mRNAs were observed, with the major forms being approximately 2.8 and 1.6 kb; the total amount of CN message was about 15% that in brain. The presence of mRNA isoforms of the catalytic subunit may provide for isoenzymes of this phosphatase having distinct phosphoprotein substrate specificities or regulatory properties. The structural relatedness of CN to other mammalian serine/threonine protein phosphatases was highest over a region of approximately 240 amino acids near the amino terminus of this subunit, with greater similarity to protein phosphatase 2A than protein phosphatase 1. The conservation of many regions found in lambda phage phosphatase (Cohen, P.T.W., and Cohen, P. (1989) Biochem. J. 260, 931-934) indicates a common origin for the catalytic domain of this enzyme. Rockville, Maryland 20852. SO - J Biol Chem 1990 Jul 5;265(19):11312-9. Neg-237. PMID:8554340 | PIR:HSBO3 TI - Successive elution by ion-exchange chromatography of H3-H4 histone complexes differing in their degree of acetylation. AB - On Biorex 70 ion exchanger at neutral pH the histones H3 and H4 are usually eluted by 4 M guanidinium chloride (gdm Cl). In order to protect cysteines and methionines from oxidation we systematically added 2-mercaptoethanol to the elution buffer. This resulted in the two histones being unexpectedly eluted together at around 1 M gdm Cl. The use of a shallower gradient resulted in a division in the peak of histones, with the acetylated species of H3 and H4 being eluted first and the nonacetylated species of H3 and H4 eluted last. When histone H3 or histone H4 was applied alone or when the chromatography was performed at low pH, these histones were eluted in the usual position at about 4 M gdm Cl. These events mean that the simultaneous elution of the histones H3 and H4 at about 1 M gdm Cl involves the formation of H3-H4 complexes. Therefore, the H3-H4 complex may be obtained by ion-exchange chromatography as the H2A-H2B complex was previously; furthermore, the former was fractionated according to postsynthetic modifications. This finding provides a new basis for explaining some of the previous elution profiles of chromatin extracts. SO - Arch Biochem Biophys 1996 Jan 1;325(1):29-38. Neg-238. PMID:2820791 | PIR:C1HURB TI - Identification of erythro-beta-hydroxyasparagine in the EGF-like domain of human C1r. AB - Previous studies [(1987) Biochem. J. 241, 711-720] have shown that position 150 of human C1r is occupied by a modified amino acid that, after acid hydrolysis, yields erythro-beta-hydroxyaspartic acid. In view of further investigations on the nature of this residue, peptide CN1a T8/T9 TL8 (positions 147-155) was isolated from C1r A chain by CNBr cleavage followed by enzymatic cleavages by trypsin and thermolysin. Amino acid analysis, sequential Edman degradation and FAB-MS of this peptide indicate that the residue at position 150 is an erythro-beta-hydroxyasparagine resulting from post-translational hydroxylation of asparagine. X, France. SO - FEBS Lett 1987 Sep 28;222(1):129-34. Neg-239. PMID:1972623 | PIR:DVHU1 TI - mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug resistance profiles. AB - Three high-level multidrug-resistant sublines of the human T-lymphoblastoid cell line CCRF-CEM were selected independently with either actinomycin D, vincristine or adriamycin. They exhibited distinct quantitative differences of cross-resistance profiles, and showed amplification and marked expression of the mdrl/P-glycoprotein gene. DNA and RNA were prepared from the cell lines, and additionally from three cell samples of patients suffering from acute lymphatic leukemia. Applying the polymerase chain reaction (PCR) for amplification, we cloned and sequenced from these sources segments of the mdrl/P-glycoprotein gene around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P-glycoprotein. Altogether, only 2 single nucleotide differences in an intron were found in 2 out of 40 recombinants each harboring a 209 bp genomic or a 269 bp cDNA fragment of the mdrl/P-glycoprotein gene. Our result does not support the idea of clustered point mutations in this segment of the P-glycoprotein gene as a cause of different multidrug resistance profiles. We additionally examined another segment of the P-glycoprotein gene in its second half, essentially delivering the same negative result, though. of Germany. SO - Biochem Biophys Res Commun 1990 Jun 15;169(2):796-802. Neg-240. PMID:2456455 | PIR:HSHUP1 TI - Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies. AB - The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed. SO - Mol Immunol 1988 Apr;25(4):403-10. Neg-241. PMID:3375238 | PIR:MMHUE4 TI - Selective expression of an erythroid-specific isoform of protein 4.1. AB - We have conducted comparative analysis of nucleotide sequences encoding erythroid and lymphoid protein 4.1 isoforms. The lymphoid protein 4.1 isoforms exhibit several nucleotide sequence motifs that appear to be either inserted into or deleted from the mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of the alternatively spliced mRNA during erythroid maturation implies the existence of a lineage-specific splicing mechanism whose activity is triggered by terminal maturation. Haven, CT 06510. SO - Proc Natl Acad Sci U S A 1988 Jun;85(11):3713-7. Neg-242. PMID:2344289 | PIR:A32541 B32541 TI - Molecular cloning of human submandibular histatins. AB - Histatins are a group of histidine-rich polypeptides found in human parotid and submandibular gland secretions. These polypeptides are microbiocidal, possibly involved in maintaining the acquired enamel pellicle, and enhance the glycolytic activity of certain oral micro-organisms. Histatins 1, 3 and 5 are homologous proteins with 38, 32 and 24 amino acid residues, respectively; the cDNAs coding for histatins 1 and 3 have now been isolated and sequenced. The cDNA sequences were highly homologous but contained differences throughout their length, indicating that they arise from different genes that may be derived from a common ancestral gene. Northern blots were hybridized to a series of oligonucleotide probes, designed on the basis of histatin cDNA sequences, and these positively identified mRNAs for histatins 1 and 3. In addition, there was a third mRNA, which hybridized to several histatin oligonucleotide probes, suggesting that histatin 5 might be derived from a distinct mRNA and not by proteolytic processing of histatin 3. A Northern blot of macaque parotid gland total RNA also showed three histatin mRNAs, indicating that similar histatins exist in a non-human primate. 02118. SO - Arch Oral Biol 1990;35(2):137-43. Neg-243. PMID:6272839 | PIR:OACH TI - Complete nucleotide sequence of the chicken chromosomal ovalbumin gene and its biological significance. AB - The nucleotide sequence of the entire chicken chromosomal ovalbumin gene has been determined. The gene is 7564 nucleotides in length to code for a mature messenger RNA of 1872 nucleotides. Comparison of the sequence at the 5'-terminal region of the gene with that reported by others has revealed multiple polymorphic nucleotides in the structural, intervening, and flanking DNA sequences. Some of the polymorphic sites occur at positions very close to splice junctions or the eucaryotic promoter sequence, yet apparently have little or no effect on the expression of this gene. The heptanucleotide promoter sequence TATATAT present in the 5'-flanking region of the ovalbumin gene does not occur within the confines of the gene. Nevertheless, multiple Hogness box sequences similar to those found in other eucaryotic genes were delineated within the boundaries of the gene. These internal Hogness box sequences are not used for transcription initiation. Similarly, the hexanucleotide sequence AATAAA common to all eucaryotic messenger RNAs at the 3'-untranslated region occurs seven additional times within the ovalbumin gene. These sites are not used for transcription termination or polyadenylation. Thus, although these sequences may play important roles in the initiation or termination of gene transcripts as well as polyadenylation of the transcripts, the specificity for such biological functions must not reside within these sequences alone. Furthermore, sequences complementary to the highly conserved rat U1 small nuclear RNA have been found throughout the gene. Many of these regions of complementarity occur in the structural sequences. If the small nuclear RNA does play a role in splicing, the specificity must be provided also by other as yet undefined components. SO - Biochemistry 1981 Oct 27;20(22):6437-46. Neg-244. PMID:6689067 | PIR:FGHUA FGHUG TI - Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen. AB - cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen. SO - Nucleic Acids Res 1983 Nov 11;11(21):7427-34. Neg-245. PMID:2920035 | PIR:A32794 TI - The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs. AB - The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein. SO - Biochem Biophys Res Commun 1989 Feb 15;158(3):646-51. Neg-246. PMID:2803315 | PIR:A33430 TI - Primary structure and functional expression of h-caldesmon complementary DNA. AB - Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon. Medical School, Japan. SO - Biochem Biophys Res Commun 1989 Oct 16;164(1):503-11. Neg-247. PMID:8248100 | PIR:KBBOA2 TI - Overproduction of bovine beta-casein in Escherichia coli and engineering of its main chymosin cleavage site. AB - A cDNA clone containing the entire coding region for bovine beta-casein A3 flanked by 53 base pairs of 5' non-coding and 358 base pairs of 3' non-coding sequences was isolated from a bovine mammary cDNA phagemid library. The coding segment for mature beta-casein was subcloned into the T7 expression system, in which the expression of recombinant beta-casein was controlled by the T7 gene 10 promoter and ribosome binding site. High level expression of Met-beta-casein to approximately 20% of the total soluble proteins was obtained in Escherichia coli within 2 h after induction of T7 RNA-polymerase synthesis. In an attempt to induce secretion the coding segment for mature beta-casein was coupled to the ompA translational initiation signal and signal peptide coding sequence but no secretion of the fusion protein and no processing of the signal peptide from the fusion protein was observed. Instead, the Met-beta-casein could be isolated in a soluble form from E.coli cells after an osmotic shock, indicative of a periplasmic location. This procedure did not lyse the cells. The protein was purified to homogeneity after a pH 4.8 isoelectric precipitation followed by reversed-phase high-performance liquid chromatography. The beta-casein cDNA was altered to change the main chymosin cleavage site in beta-casein at position 192-193 in two ways, namely from Leu-Tyr to Pro-Pro and to Leu-stop.(ABSTRACT TRUNCATED AT 250 WORDS) Research (NIZO), Ede, The Netherlands. SO - Protein Eng 1993 Sep;6(7):763-70. Neg-248. PMID:2143188 | PIR:FGHUA FGHUB FGHUG TI - A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule. AB - The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain. Philadelphia, Pennsylvania 19140. SO - J Biol Chem 1990 Aug 15;265(23):13669-76. Neg-249. PMID:2140597 | PIR:A43803 TI - Mouse vimentin: structural relationship to fos, jun, CREB and tpr. AB - We have isolated and characterized mouse cDNA clones representing the entire coding region of vimentin. RNA blot analysis of different stages during development has revealed differential control in the expression of vimentin mRNA in the different tissues studied. The nucleotide sequence extends 1800 base pairs and contains the 466 amino acid mouse vimentin polypeptide chain, flanked by 90 base pairs 5' and 312 base pairs 3' untranslated region. Conformational analysis of the deduced amino acid sequence was used to localize the three known structural domains: a non-alpha-helical N-terminal head of 81 residues, a rod-like domain of 330 residues arising from three alpha-helices, and a non-alpha-helical C-terminal domain of 55 residues. Amino acid sequence comparisons with other species revealed high sequence conservation of mouse vimentin to hamster (98.7%), human (96%), and chicken (88%) protein. Computer sequence analysis also revealed domains of significant homology between different alpha helical regions of vimentin and the DNA binding-leucine zipper domain of several proto-oncogenes and transcription regulators. Specifically, 50-70% structural similarity was observed between the basic domain of the DNA binding region of the nuclear proto-oncogene products c-fos and its related antigen fra-1, c-jun and the cAMP-responsive DNA binding protein CREB, with part of the N-terminal half region of helix 1b of vimentin. When the leucine zipper domains of all these proteins were compared to vimentin, at least two different regions of similarity in the vimentin molecule were found reaching up to 53% for jun, 60% for fos, and 76% for CREB. Further analysis revealed several domains of significant similarity (50%) between all alpha-helices of the rod domain of vimentin and the N-terminal (approximately 210 residues) activation domain tpr of the oncogenic raf. 77030. SO - Oncogene 1990 May;5(5):645-55. Neg-250. PMID:3447177 | PIR:A27335 TI - Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase. AB - The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three-dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three-dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains. Francisco 94143-0448. SO - Proteins 1987;2(3):177-87. Neg-251. PMID:8890162 | PIR:S67669 TI - Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. AB - A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics. USA. SO - EMBO J 1996 Sep 16;15(18):4884-99. Neg-252. PMID:1368637 | PIR:HHHU86 TI - Molecular cloning of cDNA encoding a human heat-shock protein whose expression is induced by adenovirus type 12 E1A in HeLa cells. AB - We have identified by differential plaque hybridization, human cDNA clones encoding a member of a heat-shock protein family (hsp 90 alpha) in the cDNA library of Adenovirus Type 12 E1A transfected HeLa cells. The complete nucleotide sequence of one of the clones (pHB76-114A) was identified. The sequence of 2912 base pairs had a single reading frame with a coding potential for an 84,672-Da protein. The amino acid sequence was highly homologous, but not identical, to that of the human hsp 90 alpha gene isolated from human peripheral blood lymphocytes [M. Yamazaki, K. Akaogi, T. Miwa, T. Imai, E. Soeda and K. Yokoyama, Nucleic Acids Res., 17, 7108 1989)]. This cDNA hybridized with RNA species which increased 5- to 20-fold upon heat shock and more than 5-fold in the differentiation stage of human Tera 2 cells. Tsukuba Life Science Center, Ibaraki, Japan. SO - Agric Biol Chem 1990 Dec;54(12):3163-70. Neg-253. PMID:6952256 | PIR:EQBOA TI - Adrenal opioid proteins of 8600 and 12,600 daltons: intermediates in proenkephalin processing. AB - [Met]Enkephalin-containing proteins of 8600 and 12,600 daltons have been isolated from acid extracts of bovine adrenal medulla and purified to homogeneity, and their sequences have been determined by a combination of automated Edman degradation, tryptic mapping, and enzymatic time-course hydrolysis. The 8600-dalton protein contains one copy of the [Met]enkephalin sequence at the COOH terminus and the 12,600-dalton protein contains three copies of [Met]enkephalin, of which two are internal and the third is at the COOH terminus. They possess identical NH2-terminal amino acid sequences, suggesting that the 8600-dalton protein is derived from the 12,600-dalton protein by intracellular proteolytic processing. This is supported by results from tryptic maps of both proteins. Furthermore, chemical analysis of the tryptic peptides obtained from the 12,600-dalton protein indicates that it also contains the amino acid sequence that corresponds to a previously characterized enkephalin-containing polypeptide of 3800 daltons (peptide F) [Jones et al. (1980) Arch. Biochem. Biophys. 204, 392-395]. All three polypeptides appear to be intermediates in posttranslational processing of a still larger polyenkephalin precursor molecule, proenkephalin, and part of a biosynthetic pathway leading to smaller enkephalin-containing polypeptides and free enkephalins. SO - Proc Natl Acad Sci U S A 1982 Mar;79(6):2096-100. Neg-254. PMID:2495817 | PIR:KKBOB TI - Specificity of milk-clotting enzymes towards bovine kappa-casein. AB - Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105. SO - Biochim Biophys Acta 1989 May 1;995(3):221-4. Neg-255. PMID:7983046 | PIR:HSHUP1 HSHUP2 TI - Characterization of a human locus in transition. AB - The spermatid-specific nucleoprotamine genes PRM1 and PRM2 and the transition protein gene TNP2 are clustered at a single site on human chromosome 16p13.2. To begin to understand the mechanism governing their genesis and coordinate regulation the primary sequence of this approximately 40.6 kilobase region was determined. This cluster of genes is embedded within a series of repetitive elements, including numerous Alu elements distributed at a frequency of > 1 Alu element/kilobase. Multiple Alu elements have integrated into separate truncated L1 sequences within this region. Many of these Alu elements are tandemly inserted or clustered. The role of repetitive elements in the genomic organization and evolution of this gene cluster is discussed. Computer-assisted sequence analysis revealed the presence of structural sequence elements often associated with the boundary regions of active transcriptional domains. Further analysis identified a CpG island at the 3' end of this segment of chromosome 16 and other candidate coding segments within this region indicative of an additional linked gene. These sequence landmarks are commensurate with the complexity of the region. Medicine, Detroit, Michigan 48201. SO - J Biol Chem 1994 Dec 9;269(49):31067-73. Neg-256. PMID:6712597 | PIR:UDCH TI - Cystatin. Amino acid sequence and possible secondary structure. AB - The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure. SO - Biochem J 1984 Feb 1;217(3):813-7. Neg-257. PMID:6304699 | PIR:FNBO FNHU TI - Isolation and characterization of cDNA clones for human and bovine fibronectins. AB - A bovine fibronectin (FN) cDNA clone (pFB1) was isolated by screening a cDNA library of calf testis fibroblasts with a synthetic oligonucleotide probe. The probe was a mixture of eight 14-base-long oligonucleotides designed from the amino acid sequence Glu-Cys-Phe-Met-Pro present in the Mr 3,000 COOH-terminal fragment of bovine plasma FN [Petersen, T.E., Thogersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L. & Magnusson. S. (1983) Proc. Natl. Acad. Sci. USA 80. 137-141]. pFB1 contained a 1,000 base-pair (bp) insert comprising the complete 3' noncoding sequence (690 bp) and approximately equal to 300 bp of the coding region. The clone pFB1 was used as a radioactive probe in the screening of a human cell line (Hs 578T) cDNA library. Eleven positive cDNA clones were detected, one of which, named pFH1, contained a 2,000-bp insert comprising the complete 3' noncoding region (693 bp) and approximately equal to 1,300 bp of the coding region of human FN. The sequences of the clone pFB1 insert and of the homologous region in clone pFH1 were determined. The nucleotide sequences are 90% homologous. Six amino acid changes were found, clustered in an area connecting two structural domains described in bovine plasma FN. Furthermore, the 204 COOH-terminal amino acid sequence of bovine FN was completed by overlapping two peptide fragments (MrS 3,000 and 23,000). Clone pFH1 was used in estimating the size of human fibronectin mRNA (7,900 bases) through blot hybridization analysis. Southern blot studies suggest that human FN is coded by a unique gene. SO - Proc Natl Acad Sci U S A 1983 Jun;80(11):3218-22. Neg-258. PMID:3476566 | PIR:SBHUP SBMQPI TI - Molecular basis of salivary proline-rich protein and peptide synthesis: cell-free translations and processing of human and macaque statherin mRNAs and partial amino acid sequence of their signal peptides. AB - Acidic proline-rich phosphoproteins and phosphopeptides are abundant components of parotid and submandibular salivary secretions in man and in the subhuman primate, Macaca fascicularis. The major acidic proline-rich proteins and the proline-rich phosphopeptide, statherin, of man and macaques have been shown to be potent inhibitors of calcium phosphate precipitation and are thought to function in the oral environment by maintaining saliva supersaturated with respect to calcium phosphate salts. Little is known about the biosynthesis of these proline-rich phosphoproteins and peptides, and the aim of the present work was to determine the structural relationship between statherin precursors and native human and macaque statherin. RNA was isolated from human submandibular gland, and poly(A+) mRNA was selected by affinity chromatography on oligo(dT) cellulose and translated in a reticulocyte lysate. Electrophoretic analysis of the translation products revealed that this mRNA directed the synthesis of a large number of polypeptides with Mrs ranging from 5000 to 70,000. Immunoprecipitates, prepared with an antiserum directed against human statherin, contained a single component with a Mr of 7800, approximately 2000 daltons larger than native statherin. Radiosequencing of the in vitro precursor of statherin in immunoprecipitates demonstrated the presence of a 19-residue signal peptide. These results suggest that statherin is derived from a unique structural gene, and does not result from proteolytic processing of a large polyprotein precursor. SO - J Dent Res 1987 Feb;66(2):462-6. Neg-259. PMID:8176739 | PIR:QRECCY TI - Magnesium binding to the bacterial chemotaxis protein CheY results in large conformational changes involving its functional surface. AB - The three-dimensional crystal structure of the bacterial chemotaxis protein CheY with the essential Mg2+ cation bound to the active site reveals large conformational changes caused by the metal binding. Displacements of up to 10 A are observed in several residues at the N terminus of alpha-helix 4 and in the preceding loop. One turn of this helix unwinds, and an Asn residue that was located inside the helix becomes the new N-cap. This supports the important role that N or C-cap residues play in alpha-helix stability. In addition the preceding beta-strand becomes elongated and a new beta-turn appears. The final effect is a significant modification of the surface relief of the protein in a region previously indicated, by genetic analysis, to be essential for CheY function. It is suggested that binding of a divalent cation to CheY could play a significant part in CheY activation and consequently in signal transduction in prokaryotes. iDesenvolupament, CSIC, Barcelona, Spain. SO - J Mol Biol 1994 May 13;238(4):489-95. Neg-260. PMID:7918659 | PIR:S50130 TI - Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12. AB - The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity. SO - Biochim Biophys Acta 1994 Oct 18;1219(2):559-62.