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)

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

Human neutrophils were found to release a 91-kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDS PAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third. Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil-activating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.
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PMID:Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. 164 57

Many studies have shown that gelatinases are secreted into the medium of cultures of various cell and tissue types, including bone cells. It is not clear, however, to what extent the culture process is responsible for inducing the expression of these proteases. In the present study, gelatinolytic enzymes were extracted directly from bone and other tissues and identified as bands of activity on SDS-PAGE enzymograms using gelatin as the substrate. Two forms of gelatinase (72-kDa and 92-kDa) were present in extracts of normal young rat bone. Yields were markedly higher from compact bone than from other tissues (blood, marrow, tendon, cancellous bone, articular cartilage, and skin). More 92-kDa than 72-kDa gelatinase was extracted from bone. The proteolytic specificity of the 92-kDa gelatinase isolated from the bone extract was shown to be similar to that reported for the enzyme isolated from tissue culture media. Native type I collagen was not cleaved but heat denatured type I collagen (gelatin) and native type IV, type V, type IX and type XI collagens were degraded. The proteolytic activity was inhibited by EDTA. The results indicate that more gelatinases can be extracted from bone tissue than from other tissues using mild extraction conditions. The cellular origin and function of these enzymes in bone remain to be defined.
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PMID:Direct extraction of gelatinases from rat bone. 822 49

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.
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PMID:Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. 862 38

The direct and indirect inhibitory potential of D-penicillamine toward human neutrophil and synovial fluid gelatinase B, a marker enzyme for disease severity in RA, was investigated. Gelatinase and plasminogen activator activities were assessed by SDS-polyacrylamide gel electrophoresis zymography. D-penicillamine significantly inhibits purified and synovial fluid gelatinase B in vitro at concentrations attainable in vivo and also blocks in vitro plasminogen activation. Protease inhibition may be a mechanism of action for D-penicillamine as DMARD.
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PMID:Human gelatinase B, a marker enzyme in rheumatoid arthritis, is inhibited by D-penicillamine: anti-rheumatic activity by protease inhibition. 892 72

Although heart attack is caused by occlusion of a major coronary artery, some patients have occlusion without heart attack because these patients have sufficient collateral circulation to provide an alternate pathway for blood supply to the myocardium at ischemic risk. The growth of new capillary vessels (angiogenesis) and enlargement of preexisting vessels play an important role in the collateral development. We evaluated the hypothesis that extracellular matrix metalloproteinase (MMP) expression is altered in coronary collateral arteries (0.5-1 mm o.d.) isolated from canine hearts 2-4 months after surgical placement of an ameroid occluder around the proximal left circumflex artery (n = 4), during the development of collateral vessels and restructuring new vessels. Histologic studies (hematoxylin and eosin, trichrome, and van Gieson stains) indicated cellular proliferation and increased collagen and elastin content in collateral vessels compared with comparable-sized unoccluded arterial segments of the left anterior descending (LAD) artery. In situ MMP activity of collateral vessels, measured using denatured collagen in the gel matrix, indicated an increase in total MMP activity in the intima of collateral vessels compared with normal LAD vessels. To further identify the type of MMP, tissue homogenates were prepared from collateral and LAD vessels and analyzed by SDS-PAGE zymography. The results suggest induction of gelatinase A and gelatinase B expression in collateral vessels compared with normal LAD tissue, when identical amounts of total protein were loaded onto each lane in the gel. Based on plasminogen-casein zymography, we observed the tissue plasminogen activator level to be increased in collateral vessels. On the basis of immunoblot and mRNA (Northern blot) analyses, we determined that the MMP-1 level was induced in collateral vessels 2 and 4 months after ameroid occlusion. In contrast with MMP-1, the level of TIMP-1 (tissue inhibitor of metelloproteinases) was decreased significantly (p < 0.001) in collateral compared with LAD vessels, suggesting a role for arterial TIMP in anti-angiogenic activity. Collectively, these results suggest that chronic occlusion of a major coronary artery induces upregulation of vascular remodeling mechanisms subserving collateral development. Increased MMP-2 activity in collaterals may be associated with decreased levels of tissue inhibitor of metalloproteinases and fibrous tissue remodeling following angiogenic and (or) adaptive responses of the myocardium to chronic ischemia.
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PMID:Temporal expression of extracellular matrix metalloproteinases and tissue plasminogen activator in the development of collateral vessels in the canine model of coronary occlusion. 896 Mar 89

Matrilysin, gelatinase A and gelatinase B are matrix metalloproteinases (MMPs) implicated in normal and pathological processes that require remodelling of the extracellular matrix. In human prostate tissue, matrilysin is synthesized in ducts surrounded by inflammatory cells, and focally in prostate carcinoma, but not in normal glands. Gelatinase B expression is restricted to inflammatory cells. Gelatinase A can be found in both benign and malignant prostate tissue. MMP activities are regulated by their transition from latent to activated forms, as well as by the presence of tissue inhibitors of metalloproteinases (TIMPs). We investigated whether matrilysin can activate progelatinases A and B in the presence of their bound inhibitors TIMP2 and TIMP1 respectively. Incubation of progelatinase B-TIMP1 complex with active matrilysin resulted in 78 and 68 kDa active forms, as measured by SDS-PAGE and enzyme activity assays. TIMP-free gelatinase B was also activated by matrilysin. In addition, activation of progelatinase B by matrilysin was demonstrated in the conditioned medium of phorbol ester-treated HT1080 cells, confirming the results obtained in the in vitro experiments. In contrast, matrilysin did not proteolytically cleave gelatinase A-TIMP2 complex, but led to a transient increase in gelatinolytic activity of the proenzyme. Matrilysin did not enhance the autocatalytic conversion of its own proform. The data presented here suggest that matrilysin participates in a proteolytic cascade and can activate gelatinases in the presence of TIMPs.
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PMID:Activation of gelatinase-tissue-inhibitors-of-metalloproteinase complexes by matrilysin. 956 Mar 29

The specialized interaction between embryonic and maternal tissue is unique to mammalian development. This interaction begins with the invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. Because of their highly specialized behavior invasive cells must attach to the extracellular matrix proteins, secrete proteinases, capable of degrading matrix, and migrate through the degraded matrix; invasion is partially dependent on the proteinase activity of the cells. The objective, therefore, was to study a vitro system to examine the mechanism(s) of trophoblast cell invasion and its relationship to proteinases. Since little is known about the actual mechanism(s) involved. The mouse trophoblast cell lines established from placentas of different gestational ages were chosen to study their invasive properties in vitro. To begin to understand the biochemical basis of this behavior, the chromogenic assay and the substrate gel technique was used to analyze the cell associated and secreted plasminogen activators. All lines secrete and synthesize both urokinase-type (uPA) and tissue-type (tPA) plasminogen activators. The most invasive line SM9-2, derived from mid-gestation (day 9) placenta showed the highest enzymatic activity in the conditioned medium (CM), whereas in cell extract (CE) SM-10 line derived from late gestation placenta had the highest PAs activity. Four forms of secreted PAs in CM were of 79, 72, 43 and 35 kDa molecular weights, whereas in CE only 79 kDa molecular weight form of PA was detected using substrate SDS-PAGE gels. Additional observations from cells cultured on Marrigel Invasion Chambers also showed secretion of PAs by noninvading and invading cells in a biphasic pattern suggest the involvement of these enzymes in the extracellular proteolysis. The expression of matrix metalloproteinase gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were examined by RT-PCR in all the lines, however MMP-9 and TIMP-1 signals were strongly expressed in SM9-2 and SM-10 lines respectively. CM and CE were characterized by gelatin zymography, and the proteinases secreted by these cells in CM were confirmed to be metalloproteinases with approximate molecular masses between 52 to 92 kDa. Proteinases secreted by noninvading and invading cells cultured on Matrigel Invasion Chambers were not identical suggesting that specialized, temporally regulated metallopro-teinases are involved in trophoblast invasion. Trophoblast cell invasion in Matrigel Invasion Chambers was significantly inhibited in all the lines by using 1, 10-phenanthroline, an inhibitor of metalloproteinases. The results indicated that mouse trophoblast cells have matrix--degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for human trophoblast invasion, suggesting a similar mechanism of implantation. Trophoblast culture system described here should be useful in studying some of the early events in human placentation.
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PMID:Mouse trophoblastic cell lines: II--Relationship between invasive potential and proteases. 962 4

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.
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PMID:A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1. 966 33

In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation.
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PMID:Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography. 982 33


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