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:3.4.21.7 (plasmin)
9,023 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

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

We have developed a procedure for the use of minislab gels to electrophoretically separate proteoglycans (PGs), large macromolecules with molecular masses greater than 2.5 million Da. Our procedure is a modification of the method of C.A. McDevitt and H. Muir (Anal. Biochem. 44, 612-622, 1971) for agarose/polyacrylamide, composite tube gels. These 1% agarose/1.2% acrylamide minigels are run at 35 mA for 75 min; bands are visualized by toluidine blue staining. The subtle size differences between the large aggregating PGs isolated from rat chondrosarcoma, bovine nasal septal cartilage, and adult bovine articular cartilage (which consists of two subpopulations) can be distinguished by their migration on these large pore gels. Chondroitin sulfate chains, added to all wells as a marker of constant mobility, ran immediately behind the dye front. The distance of migration into the gel of PGs incubated overnight with cathepsin B, carboxypeptidase A, papain, plasmin, elastase, or cathepsin G varied with the size of the cleavage products. We propose the use of this procedure for a convenient assessment of cartilage PGs and a rapid, reproducible assay for proteoglycanase activity.
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PMID:Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products. 178 94

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl)mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37 degrees C resulted in the formation of MMP-3 of Mr = 45,000 by cleaving of the His82-Phe83 bond. Since this bond is unlikely to be cleaved by these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of Mr = 45,000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of Mr = 53,000 and two minor forms of Mr = 49,000 and 47,000. The 53,000 Mr intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45,000 Mr form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of Mr = 46,000 generated by APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of Mr = 45,000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to activate proMMP-3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. 238 57

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.
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PMID:Purification of rabbit bone inhibitor of collagenase. 627 44

The precursor of matrix metalloproteinase 9 (pro-MMP-9) forms a complex with the tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule, and the N-terminal domain of TIMP-1 in the complex interacts and inhibits active MMPs. We have reported that a catalytic amount of MMP-3 (stromelysin 1) activates pro-MMP-9 (Ogata, Y., Enghild, J. J., and Nagase, H. (1992) J. Biol. Chem. 267, 3581-3584). To activate pro-MMP-9 in the complex, however, an excess molar amount of MMP-3 is required to saturate the TIMP-1 in the complex. The aim of this study was to test the hypothesis that the requirement for excess MMP-3 can be circumvented by specific destruction of TIMP-1 by non-target proteinases. We have tested trypsin, plasmin, cathepsin G, neutrophil elastase, and chymotrypsin as possible inactivators of TIMP-1 and found that neutrophil elastase inactivates TIMP-1 in the complex without significant destruction of pro-MMP-9. Once TIMP-1 is inactivated, pro-MMP-9 can be readily activated by a catalytic amount of MMP-3. These results suggest that neutrophil elastase may participate in the connective tissue destruction at the inflammatory sites not only by its direct action on matrix macromolecules but also by rendering pro-MMP-9 in the pro-MMP-9.TIMP-1 complex activable by MMP-3 as well as activating pro-MMP-3.
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PMID:Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. 762 55

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

Matrix metalloproteinases (MMPs) can degrade a number of proteins that constitute the extracellular matrix. Previous studies have shown that atherosclerotic plaques contain substantial amounts of fibrin(ogen)-related antigen, and more recently, MMPs have been identified in such lesions. The hypothesis that MMPs play a role in the degradation of fibrinogen (Fg) and cross-linked fibrin (XL-Fb) was investigated. Fibrinogen became thrombin-unclottable when treated with matrix metalloproteinase 3 (MMP-3, stromelysin 1) but not with matrix metalloproteinase 2 (MMP-2, gelatinase A). Incubation of XL-Fb clots (made with 125I-Fg) with MMP-3 resulted in complete lysis after 24 h. A D monomer-like fragment was generated by MMP-3 degradation of fibrinogen, XL-Fb, and fragment DD. Immunoreactivity with monoclonal antibody (MoAb)/4-2 (anti-gamma 392-406) but not with MoAb/4A5 (anti-gamma 397-411) suggested that a major cleavage site was within the sequence participating in the cross-linking of two gamma-chains. NH2-terminal sequence analysis of they gamma-chain of the D monomer-like fragment and of a dipeptide isolated from the MMP-3 digest of XL-fibrin identified the hydrolysis of the gamma Gly 404-Ala 405 peptide bond. These data indicate that the degradation of Fg and XL-Fb by MMP-3 is specific and different from plasmin. This mechanism of fibrinolysis might be of relevance in wound healing, inflammation, atherosclerosis, and other pathophysiological processes.
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PMID:Degradation of cross-linked fibrin by matrix metalloproteinase 3 (stromelysin 1): hydrolysis of the gamma Gly 404-Ala 405 peptide bond. 885 41

92 kDa and 72 kDa gelatinases, two neutral proteinases exhibiting elastinolytic activity and secreted as zymogens by aortic smooth muscle cells, were shown to bind to insoluble elastin. The active form of each enzyme interacted with substrate more avidly than latent form. Once bound to insoluble elastin, 92 kDa progelatinase was totally unaffected by any potential activators tested (tissue kallikrein, neutrophil elastase, plasmin, and stromelysin-1), except aminophenylmercuric acetate (APMA). Binding of 72 kDa progelatinase to insoluble elastin induced a fast autoactivation of the proenzyme followed by its inactivation. This process can be partly inhibited by tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), EDTA and a synthetic inhibitor of matrix metalloproteinases (BB-94). Such an autoactivation process was also partially observed following adsorption of 72 kDa gelatinase to elastin-derived peptides but not to gelatin. Therefore, elastin can act as a template to direct its own proteolysis by 72 kDa gelatinase; such a mechanism could be relevant to the focal elastolysis in the arterial wall during arteriosclerosis.
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PMID:Binding of 92 kDa and 72 kDa progelatinases to insoluble elastin modulates their proteolytic activation. 916 80


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