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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G-50, CM-Sephadex (A-50), Sephadex G-75 gel filtration. The optimum pH of this enzyme was 8.6--8.8, and the molecular weight determined by Sephadex G-75 gel filtration was approximately 30 000. This enzyme is active on the N-acetyl-L-tyrosine ethyl ester, N-succinyl-L-phenylalanine-p-nitroanilide (of chymotrypsin substrate) but not N-tosyl-L-arginine methyl ester, N-benzoyl-L-arginine-p-nitroanilide. Also, protease-S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 micrometer), and N-tosyl-L-phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin-like proteases in mast cells and leukocyte granules. When proteases-S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.
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PMID:Purification and characterization of a chymotrypsin-like enzyme (protease-S) in thermally injured rat skin. 43 32

Modification of hen egg-white lysozyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in presence of 4-phenylbutylamine yielded derivatives, which contained 0.6--0.7 modified residues and retained about 60% of the original activity. Kinetic studies revealed that the modified-lysozyme increases approx. 20-fold the kcat of hydrolysis of SucGly2Phe-4-nitroanilide by alphachymotrypsin, without changing the Km. The apparent dissociation constant of phenylbutylamine-modified lysozyme . chymotrypsin complex was found to be 0.03 mM and independent of substrate concentration. The accelerating effect of the modified lysozyme was also observed with other p-nitroanilide substrates of alpha-chymotrypsin. However, the hydrolysis of other substrates, acylation by active site titrant or inhibition by irreversible or competitive inhibitors were uneffected. The enhancing effect of the modified lysozyme seems to be very specific since other chymotrypsin-like enzymes, or serine proteinases except delta-chymotrypsin, were not influenced and phenylbutylamine derivatives of alpha-lactalbumin or ribonuclease were lacking any enhancing effect. Smaller, but significant enhancing effect was found also in lysozyme substituted by benzylamine, beta-phenylethylamine and tryptamine and in inactive derivatives of lysozyme substituted by phenylbutylamine. Competitive inhibitors of lysozyme such as N-acetyl-D-glucose amine oligomers, (GlcNAc)2 and (GlcNAc)3 abolished partially the accelerating effect of phenylbutylamine-modified lysozyme, indicating that the substituted group is located in the vicinity of the binding site.
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PMID:Enhancement of alpha-chymotrypsin-catalyzed hydrolysis of specific p-nitroanilide substrates by 4-phenylbutylamine derivative of hen egg-white lysozyme. 71 65

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea. In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
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PMID:Protease I from Escherichia coli. Some physicochemical properties and substrate specificity. 79 43

Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.
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PMID:In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules. 97 37

A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.
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PMID:A rapid method for purification of human granulocyte cationic neutral proteases: purification and characterization of human granulocyte chymotrypsin-like enzyme. 108 Oct 3

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.
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PMID:Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes. 131 83

Two fluorogenic derivatives of amino acids are proposed as substrates for the purpose of enzymatic assay: N-benzyloxycarbonyl-phenylalanine-4-methyl umbelliferyl ester (substrate-1) and tert-butyloxycarbonyl-alanine-4-methyl-umbelliferyl ester (substrate-II). Chymotrypsin-like (hydrolysis of substrate-1), elastase-like (hydrolysis of substrate-II) esterase activity of bovine pancreatic chymotrypsin, activities of cathepsin G and elastase from human, porcine and rat neutrophils and esterase activity of human, porcine and rat serum were assayed. Differences in the level of chymotrypsin-like and elastase-like activities of human, porcine and rat serum were established. Activities of purified elastase and cathepsin G from human and animal neutrophils were shown to have no significant distinctions.
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PMID:[Determination of esterase activity of human and animal serine proteinases using fluorogenic esters of amino acids as substrates]. 144 26

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.
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PMID:A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. 150 89

Coculture of purified murine T cells with anti-CD3 monoclonal antibody (145-2C11) results in the induction of nonspecific cytotoxic T lymphocytes (CTL) with MHC-unrestricted cytolytic activity against a range of tumor targets. Serine proteases associated with effector cell granules are among the molecules postulated to play a role in cell-mediated cytolysis. The present study examines the ability of exogenous serine protease substrates to inhibit anti-CD3-activated cytotoxic T (ACT) cell-mediated killing of P815 mastocytoma and YAC1.2 lymphoma target cells. The chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to significantly inhibit ACT cell-mediated cytolysis. In contrast, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) had little, if any, effect on ACT cell-mediated cytolysis. These effects were observed with both target cell populations. Conjugate inhibition studies performed with ATEE indicated that a chymotrypsin-like serine protease is involved in a postbinding event during cytolysis. Pretreatment of either target or effector cells with ATEE prior to cytolytic assay revealed that the chymotrypsin-like serine protease involved in cytotoxicity is of effector cell origin. Northern blot analysis of total RNA extracted from ACT cells revealed the presence of transcripts coding for CCP1 and CCP2 serine proteases known to be involved in antigen-specific CTL function, but little or no expression of the HF serine protease which has also been implicated in antigen-specific CTL killing. CCP2 exhibits chymotrypsin-like activity while HF displays trypsin-like activity. On the other hand, the CCP1 gene product has protease activity which resembles neither chymase nor tryptase activities. Thus, the level of mRNA expression for these serine proteases is consistent with our earlier observations, using the serine protease substrates, that a chymotrypsin-like serine protease but not a trypsin-like serine protease is involved in ACT cell-mediated cytolysis. "Lymphocyte panning" of ACT cells revealed abundant CCP1 and moderate CCP2 mRNA expression in CD4- and CD8+ anti-CD3-activated T cells with strong tumoricidal activity. CD8- anti-CD3-activated T cells with moderate cytolytic activity also expressed substantial levels of CCP1 and CCP2 mRNA, suggesting that both CD4- CD8- and CD4- CD8+ ACT cells participate in killing tumor targets. In contrast, CD4+ anti-CD3-activated T cells lacked both cytolytic activity and significant CCP1 and CCP2 mRNA expression. These findings are consistent with the involvement of chymotrypsin-like, as well as other, serine proteases in CTL-mediated lysis.
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PMID:Expression and utilization of chymotrypsin-like but not trypsin-like serine protease enzymes by nonspecific T killer cells activated by anti-CD3 monoclonal antibody. 153 39

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.
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PMID:Purification and characterization of a latent form of multicatalytic proteinase from fish muscle. 161 38


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