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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

On purification, active human fibroblast collagenase breaks down by an autolytic mechanism into two major forms (M(r) 22,000 and M(r) 27,000) and one minor form (M(r) 25,000). The ability of human collagenase to bind to the tissue inhibitor of metalloproteinases (TIMP) and to TIMP-2 resides mainly in the active site area of the 22,000 M(r) N-terminal domain of the molecule, but the 27,000 M(r) C-terminal domain also has a role in stabilizing these interactions. The 22,000 M(r) fragment is able to form a complex with TIMP and TIMP-2 which is stable to gel filtration in a similar manner to the whole molecule, but no such complexes are formed by the 27,000 M(r) fragment. Complex formation with the whole molecule is prevented by EDTA and by 1,10-phenanthroline demonstrating the importance of the active site; additionally TIMP and TIMP-2 will compete with a reversibly bound peptide hydroxamic acid inhibitor for the active site. The inhibition of enzyme activity by TIMP and TIMP-2 is less pronounced in the 22,000 M(r) fragment when compared to the whole molecule and a similar effect is seen with the peptide hydroxamic acid inhibitor and also with alpha 2-macroglobulin, suggesting a role for the C-terminal domain in interacting with these inhibitors. Whole molecule collagenase and the 27,000 M(r) fragment bind to type 1 collagen-Sepharose while the 22,000 M(r) fragment exhibits no such binding, suggesting that the C-terminal domain has an important role in the binding of enzyme to substrate.
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PMID:Fragments of human fibroblast collagenase: interaction with metalloproteinase inhibitors and substrates. 808 30

Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures showed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.
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PMID:Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio. 815 82

TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase. 826 Apr 95

The matrix metalloproteinase enzymes have been implicated in tumor invasion and metastasis by a series of correlative immunohistochemical studies. In addition, direct evidence for the role of these enzymes in this pathologic process comes from studies using specific metalloproteinase inhibitors to block tumor invasion and metastasis formation, both in vitro and in vivo. Synthetic oligonucleotide primers for four metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-10) and their tissue inhibitors (TIMP-1, TIMP-2) were selected, synthesized, and optimized in the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the qualitative profile of these enzymes and inhibitors in cultured human tumor cells and tumor tissues. These primers are specific and generate unique amplification products for each appropriate enzyme and inhibitor. Slight enhancement in the amplification of cDNA products was achieved by adding dimethylsulfoxide to the reaction mixture, but commercial enhancement reagents were ineffective. Using this RT-PCR method, cDNA amplification was successful with RNA from as few as 20 cultured tumor cells. The RT-PCR analysis was done on three invasive human colon adenocarcinomas and their paired adjacent normal mucosa. The results show MMP-1 and MMP-2 products in all three tumors, and MMP-2 detected in one of the three normal mucosa samples; TIMP-2 expression was present in two of three patients and awaits quantitative assessment of RT-PCR products.
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PMID:Reverse transcription-polymerase chain reaction phenotyping of metalloproteinases and inhibitors involved in tumor matrix invasion. 826 80

To more clearly define the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) within the human osteoblast (hOB) lineage, normal hOB and human osteogenic sarcoma cells possessing various levels of alkaline phosphatase (a marker of commitment to the osteoblast lineage) were treated with bone-resorbing agents to determine their effect on the production of interstitial collagenase, stromelysin, 72-kilodalton (kDa) gelatinase, 92-kDa gelatinase, TIMP-1, and TIMP-2. The results revealed that 1) normal hOB release copious amounts of 72-kDa gelatinase, TIMP-1, and TIMP-2; 2) hOB production of 72-kDa gelatinase and TIMP-2 is not regulated by agents that promote bone resorption (e.g. phorbol-12-myristate 13-acetate, recombinant human interleukin-1 beta, tumor necrosis factor-alpha, PTH, and vitamin D3); 3) normal hOB fail to secrete collagenase, stromelysin, or 92-kDa gelatinase when cultured on plastic or a type I collagen substratum, even in response to bone-resorptive agents or mononuclear cell-conditioned medium; 4) in contrast, certain of the osteogenic sarcoma cell populations produce collagenase, stromelysin, and 92-kDa gelatinase, especially when exposed to bone-resorbing stimuli; 5) in general, the capacity for metalloenzyme production by osteogenic sarcoma cell lines varies inversely with their alkaline phosphatase expression; and 6) the most committed (highest alkaline phosphatase) osteogenic sarcoma cell line, SAOS-2, precisely mimics the metalloproteinase profile of normal hOB. The results suggest that the expression of most metalloproteinases is under strict repression within the differentiated normal hOB, and cellular development is associated with diminished capacity to elaborate such enzymes.
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PMID:Expression of metalloproteinases and tissue inhibitors of metalloproteinases in human osteoblast-like cells: differentiation is associated with repression of metalloproteinase biosynthesis. 827 36

Matrix metalloproteinases (MMPs) play a role in tissue remodelling and angiogenesis. We have investigated the expression and regulation of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin), MMP-9 (gelatinase B) and their inhibitors TIMP-1 and TIMP-2 in human umbilical vein, femoral vein and microvascular endothelial cells, and compared these data with those obtained with human synovial fibroblasts. Non-stimulated vein endothelial cells expressed the mRNAs for MMP-1, MMP-2, TIMP-1 and TIMP-2. MMP-3 mRNA and protein were undetectable or only weakly expressed, but could be stimulated by the inflammatory mediator tumour necrosis factor alpha (TNF alpha). The expression of MMP-3 and MMP-1 was further enhanced by phorbol 12-myristate 13-acetate (PMA). Phorbol ester also induced TIMP-1 and MMP-9, the expression of the latter being further enhanced by TNF alpha or interleukin 1 alpha (IL-1 alpha). Similar stimulatory effects were observed in microvascular endothelial cells. Hence the inflammatory mediator TNF alpha induces/enhances the production of several matrix metalloproteinases in human endothelial cells. On the other hand, MMP-2 and TIMP-2 were not affected or were affected in a variable way by TNF alpha and/or phorbol ester, suggesting a dissimilar regulation of these proteins. The cyclic AMP-enhancing agent forskolin affected the production of MMPs in a cell-type-specific way. In human vein endothelial cells it enhanced the PMA-mediated induction of MMP-9, whereas it suppressed this induction in human microvascular endothelial cells and in synovial fibroblasts. On the other hand, forskolin suppressed the PMA-mediated induction of MMP-1 and MMP-3 in synovial fibroblasts, while it enhanced or did not affect this induction in various types of human endothelial cells. These observations may have implications for future pharmacological intervention in angiogenesis.
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PMID:Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester. 828 80

Interstitial collagenase (EC 3.4.24.7, matrix metalloproteinase-1, MMP-1) is synthesized and secreted by many cells, and plays an important role in a wide variety of pathophysiological degradation processes of extracellular matrices. The activity of MMP-1 is regulated by tissue inhibitors of metalloproteinases, TIMP-1 or TIMP-2, which form a non-covalent complex with the active enzyme. We raised monoclonal antibodies against zymogen of MMP-1, proMMP-1 purified from human skin fibroblasts. The antibodies recognized both precursor and active forms of MMP-1, but did not cross-react with 72-kDa and 92-kDa gelatinase/type IV collagenases or stromelysin-1. A specific and sensitive one-step sandwich enzyme immunoassay for human MMP-1 was developed using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The assay can be completed within 1 h (30 min for immunoreaction and 15 min for color development) and the sensitivity is 0.12 microgram/l with the linearity between 0.12 and 10 micrograms/l. Active MMP-1 shows 1.3-fold higher absorption at 492 nm than proMMP-1. However, the recognition rate of MMP-1 is decreased to approximately 50% and < 3% for the MMP-1-TIMP-1 and MMP-1-TIMP-2 complex forms, respectively. The MMP-1 levels in human sera from 120 healthy subjects are shown to be in the range of 8.5 +/- 5.2 micrograms/l (mean +/- S.D.) and the levels of 95% of the samples range from 0 to 20 micrograms/l.
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PMID:A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies. 830 49

Secretion of proteolytic enzymes by cells has been implicated in tissue remodeling during embryonic development as well as in invasive neoplastic diseases. We studied the regulation of type-IV-collagenase activity in Tera 2 human embryonal carcinoma cells, which in the undifferentiated state proliferate rapidly and are tumorigenic. The undifferentiated cells produced relatively low levels of matrix-metalloproteinase-2 (MMP-2) activity. This activity was not markedly affected by exogenous basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though the plasminogen activator activity of the cells was increased by these agents. Tera 2 cells can be induced by retinoic acid to differentiate into quiescent cells, of which many express neuronal characteristics. The type-IV-collagenase activity of the cells increased markedly during the differentiation. This increase was mainly due to increased expression of MMP-2. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was not markedly affected by the differentiation of Tera 2 cells. The results show that in the Tera 2 cell system, increased expression of MMP-2 is characteristic of the differentiated derivatives. This is in contrast with many other model systems, where increased type-IV-collagenase activity is associated with the malignant phenotype. This pattern of regulation may reflect the facts that Tera 2 cells resemble early embryonic cells and that their differentiation mimics related cell-differentiation processes in the developing embryo.
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PMID:Increased expression of the matrix metalloproteinase 2 in differentiating Tera 2 human embryonal carcinoma cells. 831 5

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

Matrixmetalloproteinases (MMP), such as type IV collagenases and interstitial collagenases, play an important role in tumor invasion and metastasis. And tissue inhibitor of metalloproteinases (TIMP) inhibit collagenolytic activity of these enzymes. We investigated the gene expressions of MMP-9 (92 kDa type IV collagenase), MMP-2 (72 kDa type IV collagenase), TIMP-1 and TIMP-2 in bladder cancers by Northern blot and slot blot hybridization. The mRNA levels of MMP-2, TIMP-1 and TIMP-2 increased in the cases with invasion and metastasis of bladder cancers. These findings suggest that MMP-2 acts as a regulator of the invasion and metastasis of bladder cancers. The MMP-2/TIMP-2 ratio increased as tumor invasion and metastasis progressed, suggesting that an imbalance in the MMP and TIMP ratio promote the invasion and metastasis of bladder cancers. And we also investigated the gene expressions of c-fos that activate the collagenase genes, and there was a correlation between c-fos and MMP-2 in gene expressions. It is suggested that fos gene may play an important role for the invasion and metastasis in bladder cancers.
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PMID:[Gene expressions of type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) in human bladder cancers]. 832 Aug 89


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