Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A widely distributed protein methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-methionine to the free carboxyl groups of D-aspartyl and/or L-isoaspartyl derivatives of L-aspartyl and L-asparaginyl residues. This enzyme has been postulated to function in the repair or the catabolism of age-damaged proteins. We present here the complete amino acid sequence of the more basic isozyme I of this enzyme from human erythrocytes. The sequence was determined by Edman degradation and mass spectral analysis of overlapping trypsin, Staphylococcus aureus V8 protease, Pseudomonas fragi endoproteinase Asp-N, cyanogen bromide, and hydroxylamine-generated fragments. The NH2-terminus is modified by acetylation and the protein contains 226 amino acids for a calculated molecular weight of 24,575. This value is in good agreement with the molecular weight determined for the purified protein by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and by gel filtration chromatography under nondenaturing conditions. The identification of 2 different amino acid residues at both positions 22 and 119 may indicate the presence of allelic variants or of two or more closely related structural genes. Finally, comparison of this sequence with those of methyltransferases for RNA, DNA, and small molecules, as well as other S-adenosylmethionine-utilizing enzymes, shows that many of these proteins share elements of three regions of sequence similarity and may be structurally or evolutionarily related.
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PMID:Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. 268 70

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.
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PMID:The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites. 277 70

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.
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PMID:Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph. 291 36

The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.
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PMID:Primary structure of the nucleotide binding domain of the Ca,Mg-ATPase from cardiac sarcoplasmic reticulum. 293 85

In oxidation-dependent cytotoxicity (ODCC), cytolytic T lymphocytes (CTL) non-specifically recognize, bind to and lyse oxidized target cells (O-TC) but the precise mechanism whereby CTL react with O-TC is far from clear (Berke, G., Immunol. Rev. 1983. 72:5). Here we present evidence that CTL/O-TC interactions are blocked by aldehyde-reactive reagents such as hydroxylamine, adipic acid dihydrazide and thiocarbohydrazide and that preformed CTL/O-TC conjugates dissociate upon reduction with NaBH4, suggesting that active aldehyde groups of O-TC rather than intercellular Schiff bases are involved in the recognition and lysis of O-TC by CTL in ODCC. The aldehydes are bound to trypsin-sensitive, non-H-2 glycoproteins that appear to be different and unique in the three different target cell lines so far examined (EL4, L1210, R1.1). In view of these and previous findings we would like to suggest that in ODCC, active aldehydes react with adjacent major histocompatibility complex and perhaps other cell-surface molecules to create a multitude of modified conformations, responsible for the "polyclonal" (nonspecific) MHC recognition and lysis of O-TC by CTL, as well as for an altered pattern of H-2 antibody binding to O-TC.
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PMID:Interaction of periodate-oxidized target cells and cytolytic T lymphocytes: a model system of "polyclonal MHC recognition". 301 6

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.
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PMID:Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus. 304 61

The immune response to p-azophenyl arsonate (Ars) in A/J mice is dominated by a cross-reactive idiotype (CRI or IdCR). IdCR+ hybridoma proteins 1F6 and 3D10 produced in a single mouse by immunization with a monoclonal anti-IdCR antibody did not bind Ars [Wysocki, L., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The preservation of idiotype coupled with lack of antigen binding in the same molecules provoked an examination of their primary structures in order to localize sites involved in binding to antigen and to anti-idiotypes. The VH sequence of antibody 3D10 was determined by Edman degradation of intact chains and fragments generated by CNBr, hydroxylamine, and o-iodosobenzoic acid cleavage, by trypsin and V8 protease digestion, and by sequence analysis of mRNA. The 1F6 VH sequence was reported previously [Smith, J. A., & Margolies, M. N. (1984) Biochemistry 23, 4726-4732]. The VL sequences of 1F6 and 3D10 were determined by Edman degradation of intact chains and peptides generated by cleavage with o-iodosobenzoic acid and digestion with trypsin and chymotrypsin. Both 1F6 and 3D10 are encoded by the same VH, VK, D, and JK gene segments as are IdCR+ Ars-binding antibodies. However, 1F6 and 3D10 employ the JH4 gene segment rather than JH2. Antibodies 1F6 and 3D10 share several somatic mutations, suggesting a common clonal origin, but manifest individual mutations as well. By comparison with Ars-binding IdCR+ molecules, the substitutions in 1F6 and 3D10 likely responsible for the lack of Ars binding are localized to the heavy chain D-JH junction and/or to a substitution in light chain CDR 3.
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PMID:Complete amino acid sequences of the heavy and light chain variable regions from two A/J mouse antigen nonbinding monoclonal antibodies bearing the predominant p-azophenyl arsonate idiotype. 310 82

The complete amino-acid sequence of L-lactate-dehydrogenase from the mesophilic Bacillus megaterium was determined. 92% of the 318 amino acids were established by sequence analysis of the N-terminus, of four CNBr fragments and of one fragment obtained by cleavage with BNPS-skatole. The primary structure was completed by sequencing overlapping fragments obtained by further cleavage of suitable CNBr fragments and BNPS fragments with either trypsin, endoproteinase Lys-C, o-iodosobenzoic acid or hydroxylamine. The C-terminal amino acids were determined by degradation with carboxypeptidase A. The sequence homology between lactate dehydrogenases from B. megaterium and those from other Bacilli is 59-61% and 35-37% to those from higher organisms. The high sequence homology among lactate dehydrogenases from Bacilli, adapted to different temperatures, allows comparative studies of the structural basis of protein thermostability.
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PMID:Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, V. The complete amino-acid sequence of the mesophilic L-lactate dehydrogenase from Bacillus megaterium. 311

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.
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PMID:NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110. 313 23

Extracellular RNase Fl1 has been purified from the culture filtrate of Fusarium lateritium. The enzyme has been obtained in the electrophoretically homogeneous state with the yield about 90% and 300 fdd degree of purification. RNase Fl1 is a guanyl specific enzyme (EC 3.1.27.3) with the specific activity on RNA 1420 units/mg of protein. The total primary structure of the RNase has been determined by the automated Edman degradation of two non-fractionated peptide hydrolysates produced by trypsin and Staphylococcus aureus protease and of the hydroxylamine cleavage products of the protein. It was shown that hydroxylamine converts the RNase Fl1 N-terminal residue, pyroglutamic acid, into the hydroxyamic acid derivative sensitive to Edman degradation. RNase Fl1 consists of 105 amino acid residues (Mr 10,852) and is a structural homologue of the Fus. moniliforme RNase F1, differing from the latter by 15 amino acid substitutions outside the enzyme active site.
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PMID:[Ribonuclease Fl1 from Fusarium lateriticum. Isolation, substrate specificity and amino acid sequence]. 314 86


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