Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.
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PMID:Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori. 2 86

The Vicia angustifolia proteinase inhibitor was incubated with p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-trypsin (EC 3.4.21.4) and a main product was isolated. The purified product was different to the first trypsin-modified V. angustifolia inhibitor. The C-terminal residues of the new derivative were arginine, which was also the C-terminal of the cleaved antitryptic site; lysine was a newly exposed C-terminal. These results suggest that the new derivative lacks the C-terminal portion of the native inhibitor, which has asparagine at its C-terminus. The liberated C-terminal peptide had the following amino acid sequence: H-Glu-Glu-Val-Ile-Lys-Asn-OH. The derivative lacking the C-terminal hexapeptide still possesses inhibitory activities against trypsin and alpha-chymotrypsin (EC 3.4.21.1), however, its antichymotryptic activity was inactivated by incubation with chymotrypsin at pH 8.0.
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PMID:Isolation and activities of the trypsin-modified Vicia angustifolia proteinase inhibitor lacking carboxyl-terminal hexapeptide. 3 67

In the outer membrane of P. aeruginosa, a protein of apparent molecular weight 8,000 (protein I) is present as a major protein. Purification and chemical analysis of protein I were carried out. This protein was purified by essentially the same procedure as for the purification of the E. coli lipoprotein, which was developed by Inouye et al. (J. Bacteriol. (1976) 127, 555--563). The amino acid composition of protein I was determined. Protein I lacks proline, valine, isoleucine, phenylalanine, tryptophan, and half-cystine. Fatty acid analysis of the protein revealed that it contained 0.89 mol of fatty acids per mol of protein. Among the fatty acids hexadecanoic acid (C16:0) was predominant. In an in vivo labeling experiment, [2-3H]glycerol was incorporated into protein I. A protein with similar mobility to protein I on urea-SDS polyacrylamide gel electrophoresis was isolated from the purified peptidoglycan of P. aeruginosa by trypsin digestion. The amino acid composition of this protein was essentially the same as that of protein I. These results indicate that the outer membrane of P. aeruginosa contains a protein analogous to the E. coli lipoprotein, although considerable differences were observed in the amino acid composition and the fatty acid content.
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PMID:Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein. 10 84

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.
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PMID:Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores. 11 74

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
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PMID:Serine protease specificity for peptide chromogenic substrates. 14 72

The synthetic thrombin-inhibitor termed No. 205 (N-alpha-dansyl-L-arginine-4-ethyl-piperidine amide) found in our laboratories was studied kinetically using synthetic peptide substrates. The following results were obtained. 1. No. 205 inhibited thrombin competively with bz-Phe-Val-Arg-pNA and the Ki value obtained was extremely small, 3.7 x 10(-8) M. 2. No. 205 also inhibited trypsin competitively with bz-Phe-Val-Arg-pNA but the Ki value obtained was far larger than that for thrombin, 1.0 x 10(-5) M. 3. No. 205 inhibited F. Xa, plasmin and urokinase only to a small extent when estimated using 2 x 10(-4) M D-Val-Leu-Lys-pNA, bz-Ile-Glu-Gly-Arg-pNA and Glu-Gly-Arg-pNA, respectively. 4. No 205 differed from APPA in its specific inhibitory spectrum for thrombin as compared to trypsin, plasmin and F. Xa. The above results indicate that No. 205 is an extremely potent and highly selective reversible thrombin-inhibitor.
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PMID:Kinetic studies on the selectivity of a synthetic thrombin-inhibitor using synthetic peptide substrates. 15 13

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.
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PMID: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. 16 50

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residues of the single polypeptide chain composed of 272 amino acids. These results showed an extensive homology with the sequence of many serine proteases of the trypsin-chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.
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PMID:N-Terminal amino acid sequence of rat tonin: homology with serine proteases. 21 93

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.
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PMID:Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives. 23 4

The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.
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PMID:Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin. 31 7


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