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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.
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PMID:Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma. 146 8

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha. 165 26

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.
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PMID:The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts. 184 73

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.
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PMID:The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells. 184 53

We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.
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PMID:Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes. 191 47

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.
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PMID:A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells. 238 70

Primitive biliary cells are known to migrate from the ductal plate into the mesenchyme during human intrahepatic bile duct development, and this migration process is essential for normal development of intrahepatic bile ducts. However, its molecular mechanism is unknown. Matrix proteinases play an important role in cell migration during cancer invasion and organ development. In this study, we therefore investigated in situ expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) during human intrahepatic bile duct development, using 32 human fetal livers. We also examined in situ expression of trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B, which are matrix proteinases and activators of MMP. MMP-1 expression was noted in the ductal plate and migrating primitive biliary cells. MMP-2, MMP-3, and MMP-9 were expressed in the ductal plate. TIMP-1 and TIMP-2 were expressed in the ductal plate and migrating primitive biliary cells. Trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B were also expressed in primitive biliary cells. These data suggest that MMP, trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B play a critical role in biliary cell migration during human intrahepatic bile duct development by degrading extracellular matrix proteins. The data also suggest that MMP inhibitors (TIMP-1 and TIMP-2) and MMP activators (trypsin, chymotrypsin, and cathepsin B) play an important role in biliary cell migration. The coordinated expression of MMP, MMP inhibitors, and MMP activators may be necessary for the normal development of human intrahepatic bile ducts.
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PMID:Expression of matrix proteinases during human intrahepatic bile duct development. A possible role in biliary cell migration. 748 84

Incubation of progelatinase B, isolated from human polymorphonuclear leukocytes, with TIMP-1 leads to the formation of the progelatinase B/TIMP-1 complex. This complex behaves like a Janus in a similar manner as we previously described for the progelatinase A/TIMP-2 complex. It shows the properties of TIMP-1 and is a better inhibitor for gelatinase A than for gelatinase B. Treatment with trypsin leads to activation of the binary complex. The activity, however, amounts only to slightly more than 10% of the activity of free gelatinase B, not complexed with TIMP-1. When the progelatinase B/TIMP-1 complex inhibits an active matrix metalloproteinase, a ternary complex is generated that after activation displays a distinct higher proteolytic activity than the active binary complex. The active binary complex cannot be transformed into the active ternary complex.
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PMID:Generation and activity of the ternary gelatinase B/TIMP-1/LMW-stromelysin-1 complex. 757 48

The processing of synovial fluids of patients suffering from rheumatoid arthritis led to the characterization of a neutral metalloproteinase with polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase activating properties. The activator exhibits a relative molecular mass of M(r) 27,000 and is an active form of stromelysin. Thus, it reacts specifically with antibodies raised against human stromelysin, splits polymorphonuclear leukocyte progelatinase in a manner characteristic of stromelysin, and is inhibited by EDTA as well as by a tissue inhibitor of metalloproteinases (TIMP-2). The activator shows a high specificity for the matrix metalloproteinases, polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase. It shows only weak hydrolysis of casein and gelatin, and it does not activate fibroblast M(r) 72,000 progelatinase. Brief treatment with trypsin does not lead to a significant change in the activator's relative molecular mass, but induces a rapid loss of its activating activity for polymorphonuclear leukocyte progelatinase, while its proteolytic activity against the synthetic substrate, N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg, is increased about 3-fold. The same tryptic treatment does not affect the activator's proteolytic activity towards casein and gelatin.
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PMID:A trypsin sensitive stromelysin isolated from rheumatoid synovial fluid is an activator for matrix metalloproteinases. 829 62

Multiple forms of metalloproteinase inhibitors were found in the serum-free conditioned medium of the EJ-1 human bladder carcinoma cell line by reverse zymography assay with gelatinase A as the indicator enzyme. Two novel forms of inhibitor with apparent molecular masses of 18 and 22 kDa on nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), together with tissue inhibitor of metalloproteinases (TIMP) and TIMP-2, were purified from the conditioned medium by a series of chromatographic steps. Structural analysis showed that the 18-kDa inhibitor is a two-chain form of TIMP-2 (tc-TIMP-2) produced by proteolytic processing, and the 22-kDa inhibitor may be a partially glycosylated form of TIMP. The purified tc-TIMP-2 was separated into a 17-kDa peptide and a small peptide of about 2.5 kDa by reducing SDS-PAGE and into four isoforms with pI 7.6, 7.3, 7.2, and 6.8 by isoelectric focusing. tc-TIMP-2 has essentially the same inhibitory activity as TIMP-2 toward gelatinase A, collagenase, stromelysin, and matrilysin. Unlike TIMP-2, however, tc-TIMP-2 does not bind to the latent precursor fo gelatinase A. Similar two-chain forms of TIMP-2 were produced by its partial digestion with trypsin or less effectively with plasmin. These results suggest that proteolytic processing of TIMP-2 plays a role in the regulation of gelatinase A activity in the extracellular matrix.
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PMID:Purification and characterization of a two-chain form of tissue inhibitor of metalloproteinases (TIMP) type 2 and a low molecular weight TIMP-like protein. 831 98


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