Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.35 (matrix metalloproteinase 9)
 2,207 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

Tenascin (TN) is a large oligomeric glycoprotein that is present transiently in the extracellular matrix (ECM) of cells and is involved in morphogenetic movements, tissue patterning, and tissue repair. It has multiple domains, both adhesive and anti-adhesive, that interact with cells and with fibronectin (FN) and other ECM macromolecules. We have studied the consequences of the interaction of TN with a FN matrix on gene expression in rabbit synovial fibroblasts. Fibroblasts plated on a mixed substrate of FN and TN, but not on FN alone, upregulated synthesis of four genes: collagenase, stromelysin, the 92-kDa gelatinase, and c-fos. Although the fibroblasts spread well on both FN and FN/TN substrates, nuclear c-Fos increased within 1 h only in cells that were plated on FN/TN. TN did not induce the expression of collagenase in cells plated on substrates of type I collagen or vitronectin (VN). Moreover, soluble TN added to cells adhering to a FN substrate or to serum proteins had no effect, suggesting that TN has an effect only in the context of mixed substrates of FN and TN. Collagenase increased within 4 h of plating on a FN/TN substrate and exhibited kinetics similar to those for induction of collagenase gene expression by signaling through the integrin FN receptor. Arg-Gly-Asp peptide ligands that recognize either the FN receptor or the VN receptor and function-perturbing anti-integrin monoclonal antibodies diminished the interaction of fibroblasts with a mixed substrate of FN, TN, and VN, but had no effect on the adhesion of fibroblasts to a substrate of FN and VN, suggesting that both receptors recognize the complex. Anti-TN68, an antibody that recognizes an epitope in the carboxyl-terminal type III repeats involved in the interaction of TN with both FN and cells, blocked the inductive effect of the FN/TN substrate, whereas anti-TNM1, an antibody that recognizes an epitope in the amino-terminal anti-adhesive region of epidermal growth factor-like repeats, had no effect. These data suggest that transient alteration of the composition of ECM by addition of proteins like TN may regulate the expression of genes involved in cell migration, tissue remodeling, and tissue invasion, in regions of tissue undergoing phenotypic changes.
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PMID:The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. 751 5

The peptide substrate specificities of two matrix metalloproteinases (MMPs), interstitial collagenase (MMP-1), and 92-kDa gelatinase (MMP-9), have been examined. Starting with the parent substrate, Dnp-Pro-Leu-Gly approximately Leu-Trp-Ala-D-Arg-NH2, four separate substrate mixtures were synthesized at subsites P2(Leu) through P2'(Trp). These mixtures contained either naturally occurring L-amino acids, D-amino acids, or either of two distinct sets of miscellaneous amino acids. Combined, these mixtures gave 88 unique substitutions at each position and, over the four subsites, represented 352 potential substrates. Optimal substrates were identified using a combined high performance liquid chromatography/mass spectrometry analysis as previously reported. The results gave an extended profile of the substrate specificities for both MMP-1 and MMP-9 at subsites P2(Leu) through P2'(Trp). Using the data obtained from the mapping, a new peptide substrate, Dnp-Pro-Cha-Abu approximately Smc-His-Ala-D-Arg-NH2 (where Dnp is 2,4-dinitrophenyl, Cha is cyclohexylalanine, Abu is alpha-aminobutyric acid, and Smc is S-methylcysteine) was designed and characterized. This peptide showed a 36-fold improvement in turnover (kcat/Km) versus the parent substrate by interstitial collagenase. In addition, some collagenase subsite specificities described here were found to be different from those previously reported. Experimental data show that the observed selectivity is dependent on the original peptide template employed, which has broader implications for substrate specificity studies.
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PMID:Characterization of the peptide substrate specificities of interstitial collagenase and 92-kDa gelatinase. Implications for substrate optimization. 780 5

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

The 72-kDa gelatinase/type IV collagenase (MMP-2) is a member of the matrix metalloproteinase (MMP) family of enzymes. This enzyme is known to cleave type IV collagen as well as degrade denatured collagens. However, native interstitial collagens are reportedly resistant to MMP-2 and are thought to be susceptible only to the interstitial collagenases MMP-1 and MMP-8. In this study we report that both human and chicken MMP-2, free of tissue inhibitors of metalloproteinases (TIMPs) are capable of cleaving soluble, triple helical type I collagen generating the 3/4- and 1/4-length collagen fragments characteristic of vertebrate interstitial collagenases. MMP-2 cleaves at the same Gly-Ile/Leu bond in the collagen alpha chains as interstitial collagenases with kcat and Km values similar to that of MMP-1. MMP-2 also is capable of degrading reconstituted type I collagen fibrils. The closely related 92-kDa gelatinase/type IV collagenase (MMP-9) is unable to cleave soluble or fibrillar collagen under identical conditions indicating that the specific collagenolytic activity of MMP-2 is not a general property of gelatinases. That MMP-2, a potent gelatinase, also can cleave fibrillar collagen provides an alternative to the proposal that two enzymes, an interstitial collagenase and a gelatinase, are required for the complete dissolution of stromal collagen during cellular invasion.
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PMID:Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. 789 Jul 17

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
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PMID:Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). 806 13

A lactose-binding lectin from rat lung (RL-29) and a related lectin from Madin-Darby canine kidney (MDCK) cells have been analyzed with the primary goal of identifying post-translational modifications. The sequences show that RL-29 and the dog lectin are homologues of a lectin designated here as L-29 and elsewhere as CBP-35, epsilon BP, Mac-2, or L-34. RL-29 has a 140-amino-acid COOH-terminal carbohydrate-binding domain, a 20-amino-acid NH2-terminal domain, and an intervening domain consisting of 11 repeating elements rich in Pro, Gly, and Tyr (R-domain). The dog homologue has 14 repeating elements in its R-domain explaining its larger size. The sensitivity of the R-domain to bacterial collagenase allowed us to isolate the NH2-terminal domain and show that the NH2 terminus was blocked by acetylation and, in the accompanying paper (Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H., and Leffler, H. (1993) J. Biol. Chem. 268, 26712-26718), that the NH2-terminal domain is phosphorylated. In addition, we unexpectedly found an endogenous component, resembling 92-kDa type IV collagenase, that co-purified with L-29 and slowly digested the R-domain. Hence, L-29 is a substrate for bacterial and tissue collagenases even though the R-domain is non-collagenous. Moreover, the co-purification suggests a non-enzymatic interaction between 92-kDa collagenase and L-29.
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PMID:Primary structure of the soluble lactose binding lectin L-29 from rat and dog and interaction of its non-collagenous proline-, glycine-, tyrosine-rich sequence with bacterial and tissue collagenase. 825 5

Two members of the matrix metalloproteinase family of enzymes, interstitial collagenase and 92-kDa gelatinase, have been implicated in the pathogenesis of rheumatoid arthritis and tumor metastasis. In order to characterize the activities of these enzymes, we have developed a fluorogenic peptide substrate which is efficiently hydrolyzed by both enzymes. This substrate was developed based on the addition of the fluorescent tag, N-methyl-anthranilic acid (Nma), to several previously synthesized substrates that had been evaluated with respect to their turnover by interstitial collagenase. One substrate, Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(Nma)-NH2, had favorable solubility characteristics, was > 98% quenched, and produced a single cleavage product, Dnp-Pro-Cha-Gly, with a high fluorescence yield with both interstitial collagenase and 92-kDa gelatinase. Since the assay depends on measurement of increases in fluorescence, the position of the Nma group also proved to be important for optimization of the fluorescence signal. The assay is free from interference by organomercurial compounds and the cleavage product has excitation and emission spectra compatible with filters commonly available on commercial plate readers. The assay has been adapted to a 96-well format and provides a rapid screening protocol for the evaluation of inhibitors of these enzymes.
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PMID:A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). 836 16

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.
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PMID:Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases. 856 49

Decorin (DCN) is a ubiquitous proteoglycan comprised of a core protein attached to a single dermatan/chondroitin sulphate glycosaminoglycan chain. It may play a role in regulation of collagen fibrillogenesis and function as a reservoir of transforming growth factor beta (TGF-beta) in the extracellular milieu. We have examined the susceptibility of DCN to five different matrix metalloproteinases (MMPs): MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin) and MMP-9 (gelatinase B). MMP-2 and MMP-3 digest DCN into seven major fragments in a similar pattern. The N-terminal sequence of the two fragments generated by MMP-2 and MMP-3 is Leu211-Lys-Gly-Leu-Asn, but that of the others is Asp1-Glu-Ala-Ser-Gly. MMP-7 cleaves DCN into three major fragments which have the N-termini Asp1-Glu-Ala-Ser-Gly, Glu2-Ala-Ser-Gly-Ile and Leu244-His-Leu-Asp-Asn. Activities of MMP-1 and MMP-9 against DCN are negligible. The values of Km for the MMPs capable of degrading DCN are very similar (10-12 microM), but the kcat/Km value for MMP-7 (30.5 microM-1.h-1) is 4.5-fold higher than those for MMP-2 and MMP-3. Incubation of a DCN-TGF-beta1 complex with MMP-2, -3 or -7 results in release of TGF-beta1 from the complex. These data indicate proteolytic degradation of DCN by MMP-2, MMP-3 and MMP-7, and suggest the possibility that, under pathophysiological conditions, the digestion by the MMPs may induce tissue reactions mediated by TGF-beta1 released from DCN in the connective tissues.
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PMID:Degradation of decorin by matrix metalloproteinases: identification of the cleavage sites, kinetic analyses and transforming growth factor-beta1 release. 914 53


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