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)

The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied. Substance P was attacked primarily at the Phe-8-Gly-9 bond by a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-acting prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopeptidase (FALGPA-peptidase). Bradykinin was cleaved at Phe-5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase. Angiotensin I was rapidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPase. All these enzymes were assumed to be cell associated and were easily extracted with a mild (0.05 to 0.1%) Triton X-100 treatment. Because it was conceivable that the hydrolysis of substance P at the Phe-8-Gly-9 bond was catalyzed by a CTLP described earlier (V.-J. Uitto, D. Grenier, E. C. S. Chan, and B. C. McBride, Infect. Immun. 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventional fast protein liquid chromatography procedures. For kinetic studies, Phe-8(4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, epsilon = 545 M-1 cm-1) was synthesized to replace substance P as a substrate in kinetic studies. In reversed-phase chromatography, both NSP and substance P gave identical results with both whole cells and the purified enzyme. The CTLP has a mass of 95 kDa, and its activity is suggested to be based on an active seryl residue, on an active imidazole group, and on an active carboxyl group but not on metal cations. The enzyme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (SAAPFNA, a typical chymotrypsin substrate) at a high rate and several proteins, such as calf thymus histone, human plasma fibrinogen, milk caseins, and gelatin. Among the substrates tested, substance P showed the highest affinity (Km = 0.22 mM) for the purified enzyme. Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/liter, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAPFNA by whole cells and the purified CTLP. The hydrolysis of NSP by whole cells and purified CTLP was slightly inhibited by chlorhexidine. The results demonstrated the versatility and the effectiveness of the outer membrane of T. denticola in occasioning a rapid breakdown and inactivation of human bioactive peptides and other peptidolytic catalyses.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides. 754 86

Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. 'Prince Melon'). The enzyme is stable over a wide pH range (4-11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha-chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z-Tyr-ONp) and benzoyltyrosine ethyl ester (Bz-Tyr-OEt) were cleaved, but Z-Lys-ONp and tosylarginine methyl ester (Tos-Arg-OMe) were not cleaved by cucumisin. Oxidized insulin B-chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C-termini of all the valine residues in the oxidized insulin B-chain molecule.
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PMID:Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase. 757 59

The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
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PMID:Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis. 763 63

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Suc, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from alpha-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250-300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1-84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.
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PMID:Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell. 781 50

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.
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PMID:Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin. 791 83

1. A kinin-inactivating chymotrypsin-like serine-endopeptidase was purified 202-fold from human urine by DEAE-cellulose chromatography, gel filtration, DEAE/HPLC chromatography and affinity chromatography. It hydrolyzed bradykinin at the Phe5-Ser6 peptide bond at a rate of 1.090 mumol min-1 mg protein-1 at pH 8.0 and 37 degrees C. The molecular weight of this endopeptidase H2, estimated by SDS-polyacrylamide gel electrophoresis and by gel filtration, was 60 kDa, and its optimum pH for bradykinin hydrolysis was near 8.5. 2. Bradykinin inactivating activity was inhibited 100% by the serine-proteinase inhibitor PMFS (1 mM) and the chymotrypsin inhibitor TPCK (5 mM). Reagents such as 2-mercaptoethanol (3 mM) and pOH-mercuribenzoate (3 mM) inhibited the enzyme by 100% and 67%, respectively. 3. Endopeptidase H2 hydrolyzes the Phe-Ser bond of peptides related to bradykinin and its activity appears to be limited to peptide chains of < or = 18 amino acid residues since it does not hydrolyze BAM 22, peptide E or kininogen. 4. The molecular size and inhibition profile suggested that endopeptidase H2 differs from the serine-proteinases previously described in rat liver, rat hepatic endothelium, rat and rabbit brain. 5. The physiological role of endopeptidase H2 may be a link between the kinin and neuropeptide systems in the control of water-electrolyte balance.
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PMID:Purification and characterization of endopeptidase H2, a kinin inactivating serine proteinase (kininase) from human urine. 822 Feb 64

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.
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PMID:Characterization of a maize root proteinase. 827 5

Proteolytic activity and activity of endogenous inhibitors of endopeptidases (using chymotrypsin and papain) were studied in the myocardium of rats with experimental ischemia during an acute phase (60 min) and within 5 days after ligation of the left descending coronary artery; effects of the beta-adrenoblocking agent propranolol and the calcium antagonist verapamil on these activities was also studied. During the acute phase of ischemia, the activity of acid proteases was increased by 30%, that of Ca(2+)-activated neutral proteases by 15-20%. At the same time, the activity of serine proteases inhibitors was decreased while the activity of thiol protease inhibitors was increased. Within 5 days of coronary artery occlusion, Lysosomal thiol-dependent endopeptidases were activated in the myocardium; a considerably higher activity of the inhibitors of serine- and cysteine-containing endopeptidases was detected. The cardioactive drugs propranolol and verapamil affected selectively both endopeptidase activity and their inhibitors.
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PMID:[Activity of proteolytic enzymes and their inhibitors in experimental myocardial ischemia]. 849 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

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm of Lactococcus lactis ssp. lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and alpha-chymotryptic hydrolysates of beta-casein (beta-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin released beta-CN f193-209, kinetic constant (Ki) of which for inhibition of PepO was 60 microM. This peptide also inhibited (Ki = 1700 microM) the 95-kDa aminopeptidase (PepN) from L. lactis ssp. lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one, beta-CN f69-97, was not degradable by PepO (Ki = 4.7 microM), while the other, beta-CN f141-163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide, beta-CN f69-84, which inhibited PepO with a Ki of 8.1 microM, was isolated from the alpha-chymotryptic hydrolysate. Peptides released from beta-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degraded beta-CN f193-209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (beta-CN f193-209, f69-97, f69-84, f141-163) were readily degraded by thermolysin.
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PMID:Peptides inhibitory to endopeptidase and aminopeptidase from Lactococcus lactis ssp. lactis MG1363, released from bovine beta-casein by chymosin, trypsin or chymotrypsin. 863 36


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