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 simple in vivo model of collagen degradation has been developed, and the effects of various agents have been tested. Type I collagen was prepared from rat skin and acetylated with either [3H]- or [14C] acetic anhydride. The radiolabelled collagen was added to sterile cotton buds and incubated at 37 degrees C to allow the collagen to form native fibrils that were firmly adsorbed to the cotton matrix. After subcutaneous implantation of the collagen-gelled cotton buds into rats, the radiolabelled collagen was progressively removed over a period of weeks by an infiltrating granuloma. Of the agents that were administered directly into the cotton buds using subcutaneously implanted osmotic mini-pumps, only the synthetic collagenase inhibitors CI-A (containing a hydroxamate moiety as a zinc ligand) and CI-C (containing a thiol moiety as a zinc ligand) were able to prevent the removal of collagen: their efficacy correlated with the level of collagenase inhibitory activity assayed in the exudate fluid sequestered within the cotton bud granuloma. Of the agents that were administered systemically, including anti-inflammatory drugs and other compounds used as therapies for arthritis, only hydrocortisone was able to inhibit the removal of radiolabelled collagen. These results suggest that, in this model, interstitial collagenase, a member of the matrix metalloproteinase family, comprised the major degradative pathway for collagen. The collagen-gelled cotton bud model is a useful test system for delineating those processes that result in collagen catabolism. In addition, the model can be used for testing agents, including those of limited or unknown systemic bioavailability, in order to discover novel therapeutic agents for preventing collagen degradation in connective tissue diseases such as arthritis.
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PMID:A simple in vivo model of collagen degradation using collagen-gelled cotton buds: the effects of collagenase inhibitors and other agents. 766 28

The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgCl2 is a moderately effective activator, while p-chloromercuribenzoate and NaOCl are poor activators. It is also shown that human matrilysin and human fibroblast-type collagenase can activate pro-HNG by a mechanism that is very similar to that of stromelysin. Initially, these proteinases hydrolyze the Glu40-Met41 bond in the propeptide domain to generate an 88 kDa inactive HNG species. Collagenase also generates a 68 kDa HNG species through hydrolysis of the Ala74-Met75 bond. Ultimately, treatment with either matrilysin, collagenase or trypsin results in the production of a 65 kDa active form of HNG that arises from hydrolysis of the Arg87-Phe88 bond. This is the same active species produced on activation by stromelysin. This cleavage site is downstream of the 'cysteine-switch' residue located at position 80 and releases it, accounting for the permanent activation of the enzyme. These results suggest that matrilysin and collagenase may be physiologically relevant activators of pro-HNG and/or other progelatinase B species. Activation by HgCl2 produces an active 68 kDa enzyme due to autolytic hydrolysis of the Ala74-Met75 bond. This species retains the cysteine switch residue; however, it is shown that it is only active in the continued presence of HgCl2. Removal of the HgCl2 restores latency, indicating that this species is reversibly activated by HgCl2, which functions by complexing the sulfhydryl group of the cysteine switch residue and keeping it dissociated from the active site zinc atom. Thus, in spite of reports to the contrary, the cysteine switch mechanism can account for the latency and activation of pro-HNG.
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PMID:Proteolytic and non-proteolytic activation of human neutrophil progelatinase B. 766 17

Tissue inhibitor of metalloproteinases (TIMP)-2 forms a noncovalent complex with the precursor of matrix metalloproteinase 2 (proMMP-2, progelatinase A) through interaction of the C-terminal domain of each molecule. We have isolated the proMMP-2-TIMP-2 complex from the medium of human uterine cervical fibroblasts and investigated the processes involved in its activation by 4-aminophenylmercuric acetate (APMA). The treatment of the complex with APMA-activated proMMP-2 by disrupting the Cys73-Zn2+ interaction of the zymogen. This is triggered by perturbation of the proMMP-2 molecule, but not by the reaction of the SH group of Cys73 with APMA. The 'activated' proMMP-2 (proMMP-2*) formed a new complex with TIMP-2 by binding to the N-terminal inhibitory domain of the inhibitor without processing the propeptide. Thus the APMA-treated proMMP-2*-TIMP-2 complex exhibited no gelatinolytic activity. In the presence of a small amount of free MMP-2, however, proMMP-2* in the complex was converted into the 65 kDa MMP-2 by proteolytic attack of MMP-2, but the complex did not exhibit gelatinolytic activity. The gelatinolytic activity detected after APMA treatment was solely derived from the activation of free proMMP-2. The removal of the propeptide of the proMMP-2* bound to TIMP-2 was also observed by MMP-3 (stromelysin 1), but not by MMP-1 (interstitial collagenase). MMP-3 cleaved the Asn80-Tyr81 bond of proMMP-2*. On the other hand, when MMP-3 was incubated with the proMMP-2-TIMP-2 complex, it bound to TIMP-2 and rendered proMMP-2 readily activatable by APMA. These results indicate that the blockage of TIMP-2 of the complex with an active MMP is essential for the activation of proMMP-2 when it is complexed with TIMP-2.
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PMID:Steps involved in activation of the complex of pro-matrix metalloproteinase 2 (progelatinase A) and tissue inhibitor of metalloproteinases (TIMP)-2 by 4-aminophenylmercuric acetate. 777 54

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.
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PMID:The enterotoxin of Bacteroides fragilis is a metalloprotease. 780 55

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.
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PMID:Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). 782 72

Collagen synthesis and degradation in normal and carbon-tetrachloride-injured male Wistar rats at early and late stages of liver fibrosis, and the potential beneficial effects of zinc supplementation on liver fibrogenesis and collagenolysis have been assessed by measuring hepatic collagen content and prolyl hydroxylase and collagenase activities. No significant changes in hepatic collagen and prolyl hydroxylase activities were observed between control rats (82 +/- 25 cpm/mg protein) and rats with induced cirrhosis (107 +/- 23 cpm/mg protein) after 4 weeks of carbon tetrachloride injury. By this time, hepatic collagenase activity was significantly lower in rats with induced cirrhosis (61 +/- 9 micro units/mg protein) than in control rats (133 +/- 31 micro units/mg protein) (p < 0.05). This result was prevented by zinc administration, since hepatic collagenase activity was similar in zinc-supplemented, carbon-tetrachloride-injured rats and normal rats (148 +/- 19 micromicrons/mg protein). After 16 weeks, all carbon-tetrachloride-injured rats had cirrhosis. Hepatic collagen content and prolyl hydroxylase activity were significantly higher in carbon-tetrachloride-injured rats than in controls. These effects were partially prevented by zinc administration, since only two of the seven zinc-supplemented, carbon-tetrachloride-injured rats had cirrhosis. Moreover, prolyl hydroxylase activity was significantly lower in zinc-supplemented injured rats (263 +/- 27 cpm/mg protein) than in the non-supplemented respective controls (389 +/- 52 cpm/mg protein) (p < 0.05). No significant changes in hepatic collagenase activity were observed at this stage of liver injury.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fibrogenic and collagenolytic activity in carbon-tetrachloride-injured rats: beneficial effects of zinc administration. 783 96

Mutants in and around the catalytic zinc-binding site of human fibroblast-type collagenase have been expressed in Escherichia coli. Replacement of each of the three zinc ligands, His-199, His-203, and His-209, in the active site sequence: VAAHEXGHXXGXXH, not only destroyed catalytic activity but also led to improper folding of the polypeptide, suggesting that this sequence also serves as a structural zinc-binding site. By comparison, mutation of His-194 immediately preceding this sequence had no measurable effect on catalytic activity or on folding. Replacement of Glu-200 in the active site yielded enzymes that either were completely inactive (E200Q) or had greatly diminished (E200D) catalytic activity. Both Glu-200 mutants, however, were fully capable of forming complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1) after reaction with organomercurials. Formation of complexes with TIMP-1 appear to require a properly folded, but not necessarily catalytically competent, active site. By contrast, complexes with alpha 2-macroglobulin form only with mutants with a catalytically competent active site. Two mutants identified in this study (E200Q and D212E) appeared to be properly folded but unable to generate any catalytic activity when exposed to either p-aminophenylmercuric acetate, trypsin, or SDS.
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PMID:Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. 792 34

Pseudo-substrate analogues of collagenase from Corynebacterium rathayii, in which the scissile peptide bond is replaced by a phosphinic moiety, were synthesized and evaluated as inhibitors of this enzyme. The phosphinic tetrapeptide, Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1), was found to be a potent inhibitor of collagenase with a Ki value of 8 nM. Increasing the length of the phosphinic-containing inhibitors from tetra- to hepta-peptide size further improves the potency of these compounds. The heptapeptide analogue, Z-Phe-Gly-Pro-Phe-psi(PO2CH2)-Gly-Pro-Nle-OMe, with a Ki value of 0.6 nM, is the most potent inhibitor reported to date for bacterial collagenases. A comparison between the phosphinic analogue Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1) and the phosphonamide peptide Z-Phe-psi(PO2NH)-Gly-Pro-Nle (2) shows that for bacterial collagenase the replacement of a CH2 by an NH group results only in a modest increase in affinity from Ki = 8 nM for compound 1 to Ki = 6 nM for compound 2. Most of the phosphorus-containing inhibitors of this series are slow- or slow-tight-binding inhibitors with second-order rate constants for association and dissociation varying respectively for the kon values from 1 x 10(3) to 26 x 10(3) M-1.s-1 and for the koff values from 3 x 10(-4) to 2 x 10(-5) s-1. Interestingly, the lower affinity of the molecule containing a D residue in the P1 position of the inhibitor, compared with the molecule with an L residue in this position, is mainly the consequence of a lower rate constant for association of these D stereoisomers with the enzyme. This study demonstrates that phosphinic peptide analogues are potent inhibitors of a bacterial collagenase. The development of new phosphinic peptides should lead to the discovery of potent inhibitors of other zinc metalloproteases. Details of how the analogues were synthesized are given in Supplementary Publication SUP 50176 (14 pages), which has been deposited with the British Library Document Supply Centre, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1994) 297, 9.
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PMID:Phosphinic peptide analogues as potent inhibitors of Corynebacterium rathayii bacterial collagenase. 794 58

We have previously reported the crystal structure of truncated human collagenase (domain II) complexed with a low molecular weight inhibitor. Attempts to crystallize full-length active collagenase (i.e. domain II + III) have been hindered by autoproteolysis at the domain II/III junction at high protein concentrations. To overcome this problem, we have generated an inactive enzyme via a H149-->L,D151-->N double substitution which displaces the non-catalytic zinc atom, and shown that the altered collagenase is unable to cleave a synthetic substrate. We have also generated an 1251-->S substitution at the domain II/III junction and demonstrate an increased resistance to proteolysis compared to wild-type collagenase.
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PMID:Specific amino acid substitutions in human collagenase cause decreased autoproteolysis and reveal a requirement for a second zinc atom for catalytic activity. 795 37

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.
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PMID:Cell-associated collagenolytic activity by group B streptococci. 796 Jan 47


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