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)

An endopeptidase was isolated from Xenopus laevis skin secretions. This enzyme, which has an apparent molecular mass of 100 kDa, performs a selective cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, or Gly) of a number of peptide hormones, including atrial natriuretic factor, substance P, angiotensin II, bradykinin, somatostatin, neuromedins B and C, and litorin. The peptidase exhibited optimal activity at pH 7.5 and a Km in the micromolar range. No cleavage was produced in vasopressin, ocytocin, minigastrin I, and [Leu5]enkephalin, which include in their sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The endopeptidase activity was inhibited by divalent cation chelators and by phosphoramidon only at high concentrations (IC50 = 50 microM), whereas it was insensitive to classical inhibitors of chymotrypsin, angiotensin convertase, and serine and cysteine peptidases, as well as carboxypeptidases. It is hypothesized that this enzyme, which is distinct from neutral endopeptidase (EC 3.4.24.11), constitutes the prototype of a family of related metalloendopeptidases that inactivate peptide substrates by cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond.
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PMID:A peptide-hormone-inactivating endopeptidase in Xenopus laevis skin secretion. 172 23

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.
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PMID:A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization. 188 19

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.
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PMID:Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids. 241 Feb 67

A neutral endopeptidase which degrades luteinizing hormone-releasing hormone (LH-RH, <GLu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GLy-NH2) has been purified 900-fold from extracts of bovine anterior pituitary. This Ca2+-independent enzyme of 83 000 molecular weight (as estimated by gel filtration) cleaves LH-RH (KM = 180 microM) at the Tyr5-Gly6-His2-Trp3 bonds. Its activity is inhibited by the SH-reactive agents N-ethylmaleimide and p-(chloromercuri)benzoate but not by the OH-reactive agent diisopropyl fluorophosphate. Hydrolysis of the fluorogenic chymotrypsin substrate glutarkyl-Gly-Gly-Phe-beta-naphthylamide by this endopeptidase could not be detected. These properties differentiate the endopeptidase from chymotrypsin and from a glutaryl-Gly-Gly-Phe-beta-naphthylamide hydrolyzing activity of high molecular weight, which has been isolated from the same tissue and also hydrolyzes internal bonds of LH-RH.
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PMID:Characterization of a nonchymotrypsin-like endopeptidase from anterior pituitary that hydrolyzes luteining hormone-releasing hormone at the tyrosyl-glycine and histidyl-tryptophan bonds. 699 98

The cyclic GMP mediated non-adrenergic non-cholinergic (NANC) relaxation in field stimulated bovine mesenteric artery and its modulation by various factors was studied. Electrical field stimulation of precontracted (Phe 2.5 microM or histamine 5 microM) bovine mesenteric arteries resulted in relaxations varying between 10-70% in different preparations. Tetrodotoxin (3 microM) completely blocked the inhibitory NANC response. Preincubation with high concentrations (100 microM-1 mM) of NG-nitro-L-arginine for 15 min. significantly reduced the relaxation induced by electrical field stimulation. Blockade of cyclooxygenases and prostaglandin synthesis by indomethacin had no effect on the relaxatory response to electrical field stimulation. Neither the alpha 2-adrenoceptor selective antagonist yohimbine (1 microM) nor the alpha 2-adrenoceptor selective agonist UK 14,304 (1 microM) had any significant effect on the electrical field stimulation-induced relaxation. Pertussis toxin (100 ng/ml) was without effect on relaxations elicited by electrical field stimulation. GTP in the concentration range 10 microM-1 mM slightly potentiated the relaxant response. N-carboxymethyl-Phe-Leu (an inhibitor of enkephalinase) or aprotinin (an inhibitor of several proteases) had no significant effect on the electrical field stimulation response. Addition of trypsin (100 U/ml) in combination with chymotrypsin (20 U/ml) significantly reduced the electrical field stimulation-induced relaxation. In the present study we have found indications for the involvement of nitric oxide and possibly also peptides in mediating the inhibitory NANC response (relaxation) in bovine mesenteric arteries.
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PMID:Involvement of nitric oxide and peptides in the inhibitory non-adrenergic, non-cholinergic (NANC) response in bovine mesenteric artery. 810 66

Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyl-leucine 4-nitroanilide or 3-carboxypropanoyl-alanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (k cat > 10(2)s(-1); k cat/Km > or = 10(6) s(-1) x M(-1)) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k cat approximately 1s(-1)-34s(-1)). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and theromlysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.
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PMID:Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases. 860 51

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.
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PMID:Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. 917 7

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were K(m) = 2.8 microM, kcat = 5.3 min-1, kcat/K(m) = 2 min-1 microM-1 and K(m) = 5.0 microM, kcat = 7.0 min-1, kcat/K(m) = 1.4 min-1 microM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specificity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.
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PMID:Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases. 949 31

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.
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PMID:Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane. 1524

Amyloid beta (Abeta) fibrils are found in the brain tissue of persons with Alzheimer's disease (AD), where they accumulate as plaques. One way to reduce the level of accumulation of Abeta in the brain and potentially treat AD is with Abeta-degrading enzymes such as neprilysin (NEP) and insulin-degrading enzyme (IDE). However, enzymatic responses and degradation mechanisms of Abeta fibrils (crystalline-state Abeta) have not been investigated, particularly with respect to how to avoid cytotoxicity of the degradation products to neuronal cells. Thus, insight into mechanisms of enzymatic degradation of Abeta fibrils would be instructive as a route to elucidating different structural features related to degradation and to cytotoxicity. We report mechanisms of enzymatic degradation of Abeta with cross-beta structures and show the series of steps involved in the digestion of Abeta microfibrils to nanospheres or nanofilaments by protease XIV or alpha-chymotrypsin, respectively. These degradation products, which contained almost the same secondary structures, exhibited different cytotoxicities, indicating that relationships between nanoassembled structures and cytotoxicity of Abeta peptides are more significant than the beta-sheet content. In addition, the enzymatic digestion at the Lys28 loop region linking the two beta-sheets in Abeta fibrils is suggested as a key target related to cytotoxicity, a feature that can be selectively targeted on the basis of the choice of protease.
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PMID:Mechanisms of enzymatic degradation of amyloid Beta microfibrils generating nanofilaments and nanospheres related to cytotoxicity. 2019 18


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