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)

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.
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PMID:Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes. 636 Jan 55

A series of new azapeptide p-nitrophenyl esters containing a variety of P1 aza-amino acid residues have been synthesized, and the reaction of these azapeptides with chymotrypsin A alpha, subtilisin BPN', subtilisin Carlsberg, and human leukocyte cathepsin G at pH 4-7 has been studied. These azapeptides were found to be very useful as active site titrants and inhibitors of serine proteases with chymotrypsin-like specificity. Stable acyl derivatives of serine proteases are formed in the reaction with azapeptides and can be used for future crystallographic investigations. The effects of changing the nature of the P1' leaving group (-ONp, -OPh, -OCH2CF3, -OEt) for these azapeptides was also investigated. N-Acetyl-L-alanyl-L-alanyl-alpha-azanorleucine p-nitrophenyl ester can be used as an active site titrant for human leukocyte cathepsin G and N-acetyl-L-alanyl-alpha-azaphenylalanine p-nitrophenyl ester is a suitable titrant for chymotrypsin A alpha, subtilisins, or cathepsin G.
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PMID:Reaction of azapeptides with chymotrypsin-like enzymes. New inhibitors and active site titrants for chymotrypsin A alpha, subtilisin BPN', subtilisin Carlsberg, and human leukocyte cathepsin G. 636 57

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
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PMID:Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. 641 24

An acidic isozyme of a chymotrypsin-like esteroprotease from the mouse submandibular gland was purified and its properties were compared with those of the basic isozymes purified previously (Takuma, T., et al. (1983) Biochim. Biophys. Acta 755, 70-75), bovine pancreatic alpha-chymotrypsin, and other chymotrypsin-like enzymes of mice. The isoelectric point of the purified enzyme was pH 4.7, and the molecular weight was estimated to be 25,000 by gel filtration on Sephadex G-100. The enzyme hydrolyzed benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) 7 times more slowly than basic isozymes did, but hydrolyzed casein as slowly as the basic isozymes did. The acidic isozyme was 40 times more sensitive to chymostatin than basic isozymes were, but 10 times less sensitive than alpha-chymotrypsin was. Moreover, acidic and basic isozymes were immunologically distinct. Chymotrypsin-like esteroproteases in the submandibular gland were antigenically unique among chymotrypsin-like enzymes in various tissues of mice.
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PMID:Acidic isozyme of a chymotrypsin-like esteroprotease from mouse submandibular gland. 643 Aug 84

Antiviral activity of rabbit and mouse fibroblast interferons was irreversibly destroyed by treatment with halomethyl ketone derivatives of phenylalanine but not by treatment with a halomethyl ketone derivative of lysine. The inactivation reaction was pH dependent, suggesting the involvement of an amino acid residue ionizing in the region of pH 7. Tryptophan and phenylalanine, known ligands of interferons, protected rabbit interferon substantially against inactivation by the chloromethyl ketone derivative of N-tosylphenylalanine. Mixed bovine brain gangliosides protected rabbit and mouse interferons against inactivation by this reagent. Although halomethyl ketone derivatives of phenylalanine were originally designed and used for affinity labeling of the active site of chymotrypsin and similar enzymes, no evidence was found for a chymotrypsin-like activity of interferons. It is proposed that halomethyl ketone derivatives of phenylalanine inactivate interferon by an affinity labeling mechanism, first binding to a hydrophobic binding site and then reacting irreversibly with a nearby nucleophilic amino acid residue, which appears to be a histidine. This conclusion implies that a hydrophobic site on interferons is necessary for their antiviral activity.
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PMID:Inactivation of interferons: halomethyl ketone derivatives of phenylalanine as affinity labels. 695 95

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.
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PMID:Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni. 704 71

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylated, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor alpha 2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N-succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7 -amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidin-agarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N-succinyl-alanyl-alanyl-prolyl-phenylalanyl-beta-naphthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.
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PMID:Sperm-surface chymotrypsin-like protease activity required for fertilization in ascidians. 751 57

The influence of biological protease inhibitors, especially aprotinin, on the production of virulent hog cholera virus in cell cultures. Production of number and size of fluorescent plaques after infection PK 15 cells with HC virus depended on properties of fetal calf sera added to the medium. By affinity chromatography on bovine alpha-chymotrypsin bound to CM-cellulose inhibitory proteins against chymotrypsin-like proteases could be eliminated from inhibiting sera. The fraction free from inhibitors did not inhibit plaque formation of HC virus in contrast to the fraction containing the eluted inhibitor(s). The inhibitory properties of fetal calf sera could be measured by a plaque inhibition test. By this test no inhibitory component could be demonstrated in 30 individual serum samples from pigs which on subsequent infection with HC virus exhibited virus growth to high titres. The serum from one pig, however, was found to inhibit HC virus plaque formation in PK 15 cells. When infected, this animal reacted with an unexpected low virus replication and mild disease symptoms. The production of virulent HC virus could likewise be inhibited by addition to the medium of the bovine protease inhibitor aprotinin; the degree of inhibition being dependent on the concentration. Virulent virus was produced again after elimination of this inhibitor from the medium. A weak inhibitory effect on plaque formation was also achieved by addition of the protease inhibitor chymostatin (3 mg/5 mg) and human alpha 1x plasma inhibitor to the medium of infected cells. The inhibitory effect appears to be very specific.
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PMID:The influence of protease inhibitors of the organism, especially bovine aprotinin, on the production of virulent hog cholera virus in tissue cultures. 751 55

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.
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PMID:Inhibitors of human heart chymase based on a peptide library. 762 13

Potato chymotrypsin inhibitor-1 (pCTI-1) was biotinylated by reaction with sulfosuccinimidyl-6-(biotinamido)hexanoate. This derivative was used as a probe on Western blots for the detection and quantitation of chymotrypsin and the detection of a chymotrypsin-like serine proteinase synthesized by ovine chondrocytes in alginate bead culture. Densitometric analysis demonstrated that there was a linear relationship between the amount of chymotrypsin electrophoresed, over the range 0.1 to 10 ng, and the intensity of the band detected on Western blots using biotinylated pCTI-1 as probe, indicating that the technique could be used for the quantification of active proteinases. The biotinylated pCTI-1 detection technique was convenient to use, reproducible, and more sensitive than zymography.
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PMID:Biotin-labeled potato chymotrypsin inhibitor-1: a useful probe for the detection and quantitation of chymotrypsin-like serine proteinases on western blots and its application in the detection of a serine proteinase synthesised by articular chondrocytes. 766 71


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