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:3.4.24.17 (MMP-3)
3,419 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

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.
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PMID:High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis. 141 2

Procollagenase M(r) 85,000 (SDS-PAGE) was purified from buffy coat to homogeneity and represents a stable single polypeptide chain forming the entire proenzyme. The procollagenase can be activated by various proteinases, e.g. trypsin, chymotrypsin, cathepsin G, kallikrein and stromelysin and by different mercurial compounds. Proteolytic conversion of the latent enzyme to the active form by chymotrypsin is accompanied by a molecular weight reduction to an apparent M(r) 64,000. This active enzyme lacks the first 79 N-terminal residues. Activation by trypsin leads to a latent intermediate of apparent M(r) 70,000, lacking 48 N-terminal residues. The active enzyme is therefore generated upon prolonged incubation with trypsin by further cleavage of 22 N-terminal residues. Another latent intermediate form with apparent M(r) 69,000 is generated from the proenzyme upon incubation with leukocyte elastase by N-terminal cleavage of 53 or 64 residues, respectively. However, latent collagenase cannot be activated by plasmin. Activation by different mercurial compounds finally results in the formation of active collagenase with apparent M(r) 64,000. In contrast to the proenzyme, active collagenase can autolyse to give active M(r) 57,000 and 45,000 intermediates and two M(r) 28,000 fragments. Purification of latent leukocyte gelatinase yields three final products with apparent M(r) 98,000, 125,000 and 220,000 (SDS-PAGE; non reduced). Upon reduction, only the M(r) 98,000 form can be detected. The latent gelatinase can be activated in a similar manner as collagenase. Proteolytic activation by trypsin leads after N-terminal cleavage to an active gelatinase with sequence homology to leukocyte collagenase.
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PMID:Latent collagenase and gelatinase from human neutrophils and their activation. 148 34

Metalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme. This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium. Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells. The major inhibitor, which showed a single protein band with a molecular weight (Mr) of 21,000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metalloproteinases-2) by the analysis of its N-terminal amino acid sequence. The other was immunologically identified as TIMP. Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), Mr 72k gelatinase, and interstitial collagenase with 1:1 stoichiometry. When the latent precursor form (Mr 57k) of transin was incubated with p-aminophenylmercuric acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (Mr 45k) into the mature enzyme (Mr 42k). This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme. TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices.
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PMID:Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion. 166 1

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.
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PMID:Human 92- and 72-kilodalton type IV collagenases are elastases. 185 Apr 24

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.
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PMID:Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. 199 67

Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC50 of 0.27 microgram/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in the ELISA (IC50-0.5 microgram/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC50-700 and 215 micrograms/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC50-21 and 469 micrograms/ml, respectively) than undegraded aggrecan (IC50-0.27 microgram/ml).
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PMID:Sensitivity of monoclonal antibody, 5-D-4, for the detection of aggrecan, aggrecan fragments, and keratan sulfate. 750 1

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH2-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH2-terminal sequence of Tyr78-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to approximately 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH2 terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to approximately 6.5 fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the Gln80-Phe81 bond of proMMP-1. MMP-7 can also activate proMMP-9 up to approximately 50% of the full activity with a new NH2 terminus of Leu16-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.
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PMID:Matrix metalloproteinase 7 (matrilysin) from human rectal carcinoma cells. Activation of the precursor, interaction with other matrix metalloproteinases and enzymic properties. 789 11

alpha 1-antitrypsin, the primary physiologic inhibitor of human leukocyte elastase, is proteolytically inactivated by several matrix metalloproteinases including interstitial collagenase, stromelysin and 92 kDa gelatinase. In this report, we describe the catalytic effects of matrilysin, a recently identified metalloproteinase, upon alpha 1-antitrypsin. Matrilysin was found to be approximately 30-fold more effective than 92kDa gelatinase, 70-fold more effective than collagenase, and 180-fold more effective than stromelysin. Cleavage of alpha 1-antitrypsin by matrilysin produced two fragments of approximately 50 kDa and 4 kDa. The single cleavage occurred at the Phe352-Leu353 peptide bond, a locus within alpha 1-antitrypsin's active-site loop. These results suggest that apart from its activity against extracellular matrix, matrilysin provides a mechanism for the regulation of leukocyte elastase activity through its capacity to degrade alpha 1-AT.
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PMID:Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of alpha 1-antitrypsin. 798 May 22

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.
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PMID:Comparison of the proteoglycanolytic activities of human leukocyte elastase and human cathepsin G in vitro and in vivo. 814 41


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