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)

Katsuwokinase (KK) is a unique fibrinolytic enzyme recently found in skipjack "Shiokara," a Japanese traditional salt-fermented food. A crude enzyme extracted from skipjack Shiokara (Katsuwonus pelamis) showed a very strong fibrinolytic activity above 45 CU/g (fibrin plate method) based on plasmin. KK not only hydrolyzed fibrin but also several synthetic amido substrates, particularly pyro-Glu-Gly-Arg-pNA. The fibrinolytic activity of KK was not affected in the presence of 10% NaCl, was stable in the pH range from 1 to 10 at 37 degrees C for 30 min, and was inhibited by DFP, SBTI, BPTI, and aprotinin but not by epsilon-amino-n-caproic acid and t-4-amino-methylcyclohexane carboxylic acid. The crude enzyme contained at least four kinds of KK, and the major form purified had a pI value of approximately 5.0 and a molecular weight of 35,000. The N-terminal amino acid sequence of 21 residues, I-V-G-G-Y-E-Q-Z-A-H-S-Q-P-H-Q-V-S-L-N-S-G-, had 80% homology with that of trypsin. The fibrinolytic activity of the purified enzyme was approximately 2.6 times greater than that of plasmin by molar ratio, demonstrating its identity as a new and very potent fibrinolytic enzyme.
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PMID:A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara," a Japanese traditional fermented food. 852 30

1. Ovomucoids were purified from Muscovy duck, domestic duck and domestic goose. 2. Peptide maps of cyanogen bromide-cleaved ovomucoids from Muscovy duck and domestic duck were very similar to one another, but differed from that of goose. 3. Muscovy duck ovomucoid showed the same protease inhibitory pattern as ovomucoid from domestic duck, inhibiting trypsin in the molar ratio of 1:2 and chymotrypsin 1:1. 4. Inhibitory complexes could be detected between chymotrypsin and ovomucoid from both Muscovy and domestic duck, but not from goose, by using non-denaturing gels. 5. No complexes could be detected between DFP-inactivated chymotrypsin and any of the ovomucoids. 6. The results show that of ovomucoid from Muscovy duck more closely resembles that from domestic duck than goose.
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PMID:Comparative study of ovomucoid isolated from Muscovy duck (Cairina moschata), domestic goose (Anser anser) and domestic duck (Anas platyrhynchos). 915

1. A novel myofibril-bound serine proteinase (MBP) has been purified from ordinary muscle of the carp Cyprinus carpio. 2. It was solubilized from the myofibril fraction with acid treatment (under the conditions of 0.6 M KCl, pH 4.0), then purified by column chromatographic steps on Ultrogel AcA 54, and Arginine-Sepharose 4B. 3. The purified enzyme revealed a single protein band on SDS-PAGE, and its molecular mass was estimated to be 30 kDa by SDS-PAGE and gel filtration. 4. The optimum pH and temperature of the enzyme were 8.0 and 55 degrees C, respectively, when Boc-Phe-Ser-Arg-MCA and casein were used as substrates. 5. The enzyme hydrolyzed Boc-Gln-Arg-Arg-MCA most rapidly, and also hydrolyzed the substrates for trypsin-type proteinase, but not for chymotrypsin. The enzyme was inhibited by serine proteinase inhibitors such as DFP, STI and leupeptin. These results suggested that the enzyme was a trypsin-type serine proteinase. 6. Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the purified enzyme was reduced by addition of NaCl, but the caseinolytic activity and Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the partially purified enzyme were activated by NaCl.
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PMID:Purification and characterization of myofibril-bound serine proteinase from carp Cyprinus carpio ordinary muscle. 915 82

In this study, using zymogram analysis two proteolytic activities were identified in the mouse sarcoma 180 (S-180) cells that were activated by trypsin treatment and inhibited by both BBI and ACTI. These enzymes, with molecular weights of 46 kDa (dominant band) and 62 kDa (minor band), were mainly localized in the cytosol, and had optimal activity at pH 7 and 8 respectively. Their inhibition by DFP, BBI and ACTI but not EDTA and TPCK indicated they were trypsin-like serine proteases and may be the intracellular target-enzymes of protease inhibitors. The level of the precursor of the 62 kDa protease was significantly increased in the S-180 solid and soft tumors, whereas the level of the 46 kDa precursor was almost undetectable, implying that a physiological role may be played by these serine proteases during tumor invasion.
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PMID:Proteolytic activities of mouse sarcoma 180 cells that are inhibited by Bowman-Birk and Kunitz protease inhibitors. 928 64

His397 was replaced with alanine by site-directed mutagenesis of the cloned PRC1 gene in order to confirm the role of this residue in the proton-relay system of carboxypeptidase Y (CPY). The expressed and purified H397A showed a CD spectrum almost identical to that of the wild type enzyme, but its heat stability and conformation on heating differed somewhat. Kinetic analysis showed that the kcat values of the purified H397A toward the peptide substrates, Z-Phe-Leu and Z-Gly-Phe, were reduced to approximately 4 x 10(-5)-fold, whereas the Km values remained almost unchanged. The activity of the H397A preparation with the ester substrate, Ac-Phe-OEt, was negligible. The low activity of our H397A was lost on treatment with DFP and Z-Phe-CH2Cl, site-specific inhibitors, respectively, for Ser146 and His397, and with the HgCl2 and PCMB, SH-reagents for Cys341. After treatment with these inhibitors, the kcat value for the H397A preparation toward Z-Phe-Leu decreased 1 x 10(3)-fold or more. The value was approximately 10(-8) for the wild type enzyme. This level of activity is 10(3)-fold lower than the reported value for the same mutant of CPY [Carlsberg Res. Commun. 54, 165-171 (1989)], and more than 10-fold lower than the values for the corresponding His-to-Ala mutants of trypsin [J. Am. Chem. Soc. 114, 1784-1790 (1992)] and subtilisin [Nature 332, 564-568 (1988)]. These findings, together with the pH profiles and chromatographic behavior, are evidence that the low activity of the H397A preparation is due to contamination by wild type CPY. The decreased kcat value of our H397A mutant is the lowest reported among the corresponding histidine mutants of serine proteases. We conclude that the proton-relay system composed of Ser146 and His397 is the sole catalytic center of CPY, and that its destruction leads to complete inactivation.
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PMID:Proton-relay system of carboxypeptidase Y as a sole catalytic site: studies on mutagenic replacement of his 397. 968 40

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.
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PMID:Protease in normal human epidermal keratinocytes. 970 64

The amidine-containing alpha-aminoalkyl phosphonofluoridate 3 (Cbz-(4-AmPhGly)P(OPh)(F)) is a very potent inhibitor of trypsin-like enzymes. It was prepared by hydrolyzing the corresponding phosphonate diphenyl ester 4 followed by reaction of fluoride with the phosphonochloridate prepared from the intermediate phosphonic acid monoester 5. Compound 3 is the most potent amidine-containing organophosphorus inhibitor yet reported for trypsin-like enzymes. It inhibits trypsin and thrombin with second-order rate constants (Kobs/[I]) of 2.6 x 10(5) M-1 s-1 and 1.0 x 10(5) M-1 s-1, respectively, showing a 130-fold and a 1250-fold rate enhancement over the corresponding diphenyl ester (4). It also inactivates trypsin 2 orders of magnitude more potently than simple phosphonofluoridates such as DFP,1 Sarin and Soman. The phosphonofluoridate 3 does not inhibit other serine proteases such as porcine pancreatic elastase (PPE) and the esterase acetylcholinesterase (AChE). The phosphonofluoridate 3 is hydrolyzed rapidly in buffer solution and has a t1/2 of 4.5 s at pH 7.5.
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PMID:Synthesis and kinetic studies of an amidine-containing phosphonofluoridate: a novel potent inhibitor of trypsin-like enzymes. 983 6

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.
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PMID:Identification of serine esterases in tissue homogenates. 1003 48

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.
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PMID:Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects. 1038 Jun 52

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymatic activity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of trypsin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permit analysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDS-PAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretreatment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.
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PMID:Inhibition and labeling of enzymes and abzymes by phosphonate diesters. 1082 63


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