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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.
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PMID:Functional constituents of the active site of human neutrophil collagenase. 301 Aug 66

A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.
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PMID:A convenient fluorescent assay for vertebrate collagenases. 301 20

A novel gelatin-binding 21 kDa protein was identified in the culture medium of fibroblastic and sarcoma cells by affinity chromatography on gelatin-Sepharose. Its affinity for gelatin was lower than that of the other gelatin-binding proteins, fibronectin and the 70 kDa protein, as judged by stepwise elution by urea and arginine. The protein bound also to spermine and to some extent to heparin but not to staphylococcal protein A, bovine serum albumin, concanavalin A or plain Sepharose 4B. In gel filtration chromatography the protein eluted in fractions differing from those of fibronectin and the Mr 70,000 protein and retained its ability to bind to gelatin-Sepharose, indicating that the binding was not mediated by the two other gelatin-binding proteins. It contains intrachain disulfide bridges, as judged by analysis under nonreducing and reducing conditions. The protein is composed of two major subtypes with pI values of 5.85-6.10 and 6.55-6.75. It was sensitive to trypsin but not to collagenase or thrombin. Antiserum was raised in rabbits against the gelatin-binding proteins isolated from serum-free conditioned fibroblast culture medium. The antiserum reacted with fibronectin, the Mr 70,000 protein and the Mr 21,000 protein in immunoprecipitation experiments. Absorption of the antiserum with human plasma fibronectin did not decrease its reactivity with the Mr 70,000 and 21,000 proteins. However, absorption with the Mr 70,000 protein abolished also the reactivity against the Mr 21,000 protein, suggesting immunological cross-reactivity. The protein was synthesized independently from the Mr 70,000 protein, as shown by pulse-chase labeling experiments of cells. The production of the Mr 21,000 protein in cultured cells was enhanced by transforming growth factor-beta.
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PMID:Characterization of a novel gelatin-binding 21 kDa protein secreted by cultured adherent cells. 301 29

Tissue pieces from the wall (i.e. tunica albuginea with adjacent theca externa) of human follicles were incubated with and without various hormones and their potential influence upon the collagenolytic activity was evaluated. Following incubation the collagenase activity was determined in the incubation medium by measurement of the hydrolytic activity against the synthetic peptide 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH. Stimulated collagenolytic activity was seen in the presence of relaxin and oxytocin whereas prostaglandin E2, prostaglandin F2 alpha, progesterone and 17 beta-estradiol were without effect. It is concluded that the stimulated collagenolytic activity induced by relaxin and oxytocin may be of importance for the degradation of collagen which occurs prior to follicular rupture.
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PMID:Hormonal effects on collagenolytic activity in the isolated human ovarian follicular wall. 301 21

New synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3' subsite of the enzyme. Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3' position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3' appears relatively limited. The compound (HS-CH2-CH2-CO-Pro-Arg), which carries an arginine residue in the position P3' and had the highest inhibition constant of the series tested (Ki = 0.5 microM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS-CH2-CH2-CO-Pro-Asp) which carries an aspartic residue in position P3' and had a Ki = 70 microM. The present work revealed that the charged groups in the P3' position play a key role in the interaction of the inhibitors with the enzyme.
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PMID:New thiol inhibitor of Achromobacter iophagus collagenase. Specificity of the enzyme's S3' subsite. 302 59

Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I-collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma-anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH.
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PMID:Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase. 302 41

The zona pellucida (ZP) from pig oocytes was isolated using two different methods. In the first method, the ZP was isolated using sieving procedures. In the second method, an enzymatic step with collagenase was used in addition to sieving procedures. Several commercial collagenase preparations were tested. The macromolecular composition of the ZP isolated by these two methods was determined by two-dimensional polyacrylamide-gel electrophoresis after disulfide bond reduction. The ZP prepared by the sieving method contained four glycoprotein families with apparent molecular weights of 25,000, 55,000, 65,000, and 90,000. The ZP obtained using the enzymatic method was distinctly different, lacking the highest molecular-weight family (90,000) and containing at least three new constituents with apparent molecular weights of 70,000, 40,000, and less than 25,000. Commercial collagenase preparations, when analyzed by two-dimensional polyacrylamide-gel electrophoresis to assess homogeneity, contained numerous protein components. The trypsin-like protease concentration in the collagenase preparations was determined to be 3.4-42 X 10(-8) M as determined by activity measurement using benzoyl-DL-arginine-beta-naphthylamide as substrate or the active site titrant p-nitrophenyl-p'-guanidinobenzoate. Thus, the ZP prepared by the enzymatic method, using collagenase preparations, had an altered macromolecular composition, thereby rendering the ZP unsuitable for most structural, immunological, or sperm-binding studies.
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PMID:Proteolysis by collagenase preparations alters the macromolecular composition of the porcine zona pellucida. 302 48

Human skin collagenase is secreted by cultured fibroblasts in a proenzyme form and can be activated to a catalytically competent enzyme by a number of processes. All modes of activation studied lead to conversion of the proenzyme to a stable 42-kDa active enzyme, concomitant with removal of an 81-amino acid peptide from the amino-terminal end of the molecule. The sequence of events leading to the formation of this enzyme form has been determined by analyzing the primary structure of the conversion intermediates. Trypsin-induced activation of procollagenase occurs as a result of the initial cleavage of the peptide bond between Arg-55 and Asn-56, generating a major intermediate of 46 kDa. Treatment of the proenzyme with organomercurials, which have no intrinsic ability to cleave peptide bonds, initially results in activation of the enzyme without loss of molecular weight. This is followed by conversion to two lower molecular weight species of 44 and 42 kDa, the latter corresponding to the stable active enzyme form. The final cleavage producing this form of collagenase is not restricted to a single polypeptide bond but can occur on the amino-terminal side of any one of three contiguous hydrophobic residues, Phe-100, Val-101, Leu-102. The data suggest that both trypsin and organomercurials activate procollagenase by initiating an intramolecular autoproteolytic reaction resulting in the formation of a stable 42-kDa active enzyme species.
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PMID:The activation of human skin fibroblast procollagenase. Sequence identification of the major conversion products. 303 47

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.
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PMID:Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site. 303 60

Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation.
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PMID:Inhibition of the activation of Hageman factor (factor XII) by complement subcomponent C1q. 303 61


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