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)

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.
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PMID:Recombinant porcine collagenase: purification and autolysis. 784 Jun 5

We studied the gelatinolytic activity, caseinolytic activity and the level of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the sclera from experimentally induced axially elongated eyes. The animal models were made by the injection of 250U alpha-chymotrypsin into the posterior chamber of young albino rabbits. Animals were killed with overdoses of intravenous pentobarbital and eyes were enucleated gently. Sclera from the equatorial area was then subjected to organ culture. The enzyme activity in the media was investigated by zymography using gelatin- or casein-containing gels together with an image analyzer system. TIMP-1 levels in each medium were measured by enzyme immunoassay (EIA). All samples showed three major bands of gelatinolytic activity at molecular weights of 87, 61, and 57 kDa. Gelatinolytic activity of 87 kDa increased 1.1 to 2.9 times compared with control sclera. All of the caseinolytic activity (87, 82, 52, and 50 kDa) was also elevated 1.8 to 6.6 times in samples from experimental eyes. TIMP-1 levels were mildly increased (up to 1.41 times) in the experimental eyes. These data suggest that degradation processes might be accelerated in the sclera from experimentally induced elongated eyes.
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PMID:[Changes of expression in matrix metalloproteinase and its inhibitor level in the sclera from axially elongated eyes]. 788 24

One of the most consistent observations in abdominal aortic aneurysm (AAA) disease is the disorganization and disruption of elastin and other matrix components of the aortic wall. The enzymatic basis for the biochemical features of AAA has been investigated beginning with the demonstration on substrate gel enzymography of a typical "profile" of proteinase activities in AAA tissue extracts which degrade gelatin, casein and elastin. A recombinant TIMP-1 affinity column was developed and three of the elastolytic/caseinolytic activities with approximate molecular weights of approximately 80 kDa, approximately 50 kDa and approximately 32 kDa were partially purified from these extracts. Affinity for rTIMP-1 suggests that these enzymes are members of the matrix metalloproteinase (MMP) family. High molecular weight forms of two MMPs, collagenase (MMP-1) and stromelysin-1 (MMP-3), were also isolated from the AAA tissue on this column; active forms of MMP-1 could be demonstrated by immunoblotting techniques in this preparation under reducing conditions. Infiltrating inflammatory cells are known sources of these proteolytic activities; analysis of these cell populations in the aneurysmal aortic wall using fluorescence-activated cell counting revealed a fifty-fold increase in macrophages (a well-known source of matrix-degrading enzymes) as well as a significant increase in lymphocytes.
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PMID:Matrix metalloproteinases in abdominal aortic aneurysm: characterization, purification, and their possible sources. 795 5

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveolus-like structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominantly in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.
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PMID:Extracellular matrix and mouse mammary cell function: comparison of substrata in culture. 798 41

In addition to producing matrix degradation for normal tissue remodeling and repair, matrix metalloproteinases (MMPs) are also involved in various pathologic processes. MMPs and the tissue inhibitor of MMPs (TIMP) were investigated in primary cultures of pig fibroblasts from radiation-induced dermal fibrosis and compared to normal dermal fibroblasts. The free gelatinolytic, collagenolytic, and caseinolytic activities secreted into the culture medium were evaluated against specific 3H denatured collagen type I, native helical collagen, and casein alpha, respectively. The 72- and 68-kilodalton (kDa) forms of type IV collagenase were investigated by protease zymography and quantified by semi-automated image analysis. Transcription of the interstitial collagenase (MMP-1) and TIMP genes was studied by Northern hybridization analysis. Results revealed that in fibrotic fibroblasts, the amount of MMP-1 mRNA was greatly reduced to undetectable levels whereas the amount of TIMP mRNA was increased fourfold compared to controls. Functional assays using specific 3H substrates demonstrated an overall decrease in free MMP activities. Concomitantly, catheptic collagenolytic activity decreased in fibrotic fibroblast extracts compared to controls. These results indicate that in addition to accumulating large amounts of collagen, proteoglycans, and fibronectin, pig fibroblasts from radiation-induced dermal fibrosis also promote connective tissue matrix formation by repressing MMP-1 and stimulating TIMP expression at the transcriptional level, and by reducing overall free MMP and catheptic collagenolytic activities at the post-transcriptional level. In contrast, enzymography assays and automated image analysis demonstrated no significant change in the 72-kDa type IV collagenase activity of fibrotic pig skin fibroblasts. This opposite regulation of 72-kDa collagenase type IV to that of MMP-1 seems to indicate that it has a specific role in remodeling the extracellular matrix during wound healing, fibrogenesis, and angiogenesis.
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PMID:Expression of 72-kDa gelatinase (MMP-2), collagenase (MMP-1), and tissue metalloproteinase inhibitor (TIMP) in primary pig skin fibroblast cultures derived from radiation-induced skin fibrosis. 800 59

Matrix metalloproteinases (MMPs) together degrade virtually all the components of the extracellular matrix and are likely to play a role in remodeling of endometrial tissue during the normal menstrual cycle. Primary cultures of human endometrial stromal cells secreted a number of MMPs. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were measured in culture medium by specific enzyme assays. Production of the enzymes did not correlate with the time of the menstrual cycle at which the tissue was collected. Identities of MMP-1 and MMP-3 were confirmed by Western blots, by comparison of mol wt with those of purified enzymes on casein zymography, and by inhibition of these activities with EDTA and 1,10-phenanthroline. Northern analysis demonstrated specific messenger ribonucleic acid for pro-MMP-1 and pro-MMP-3 in phorbol myristate acetate-stimulated stromal cells. Two gelatinases were detected by gelatin zymography: MMP-2 (gelatinase-A) was present in two forms (72 and 67 kilodaltons), and MMP-9 (gelatinase-B) was present as a homodimer with a mol wt of approximately 180 kilodaltons. MMP-9, but not MMP-2, secretion was stimulated by phorbol myristate acetate. All enzymes could be activated in vitro by (4-aminophenyl)mercuric acetate. Both interleukin-1 alpha and tumor necrosis factor-alpha stimulated the secretion of MMP-1, MMP-3, and MMP-9, but not MMP-2, from the cells in a concentration-dependent manner. MMP production by endometrial stromal cells has a potentially important role in the processes of menstruation and implantation.
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PMID:Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha. 804 73

The enzymatic activities of uPA, and a collagenase-like proteinase in the post-nuclear fraction of cell homogenates of a metastatic carcinomatous cell line following X-ray irradiation were examined by the use of chromogenic substrates and by casein- or gelatin-containing zymographies and electrophoretic gel stained with avidin-conjugated peroxidase. Enhanced activities were observed in these cells, while those of 5'nucleotidase and Na(+)-K(+)-ATPase were attenuated. A partial purification and characterization of the collagenase showed that it was able to hydrolyze the heat-denatured type-I collagen more efficiently than the native one. The activation of both uPA and collagenase enables an efficient degradation of matrix barrier proteins. These findings suggest that following a certain dose range of X-ray irradiation, tumor cells may increase their ability to migrate and invade through the enhancement of uPA and collagenase activities.
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PMID:The concomitant augmentation of urokinase-type plasminogen activator and collagenase-like proteinase activities in X-ray irradiated cells of a human metastatic carcinomatous line. 809

We have set up a quantitative and sensitive enzymatic assay for proteases of different classes acting on proteoglycans, casein, or gelatin. Radiolabeled substrates were covalently attached to insoluble microcarriers and assays were performed in 96-well plates. Protease activities were determined by the release of labeled degradation products. Time- and dose-response curves were linear when the solubilization of labeled substrates did not exceed 15-20% of the initially bound molecules. Results were compared to those from zymographic analyses on proteoglycan-, gelatin-, and casein-polyacrylamide gels, as well as to the results obtained with conventional assays using soluble [3H]-casein and [3H]gelatin. Our assay procedure was more sensitive than other available methods: it detected picogram amounts of trypsin as well as picogram or nanogram amounts of the purified human matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9, depending on the specific activities of these MMPs on the different substrates. Our new procedure was appropriate for assaying the MMPs present in crude culture media conditioned by chondrocytes cultivated under various conditions.
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PMID:An assay for matrix metalloproteinases and other proteases acting on proteoglycans, casein, or gelatin. 812 75

Isolated equine blood and articular cells were investigated for proteolytic enzyme production by means of gel filtration and analysis on 14C-acetylated collagen and casein substrates. Significant amounts of collagenase and caseinase activity were produced by cultured synoviocytes stimulated with equine interleukin 1, although large amounts of collagenase also originated from neutrophils.
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PMID:Cellular sources of proteolytic enzymes in equine joints. 814 63

This study reports the effects of Simplex bone cement powder (BC) on the proliferation and production of bone resorbing factors in vitro by human adherent monocytes/macrophages. Adherent peripheral blood cells were isolated from seven healthy individuals and exposed to a dispersion of BC powder (1 mg/mL), phytohemagglutinin (PHA, 40 micrograms/mL), or medium alone at different periods of cell incubation (days 0-2, 0-7, 5-7, or 10-12). Cell proliferation was quantified by incorporation of 3H-thymidine uptake. Culture supernatants were evaluated for levels of interleukin 1-like activity (IL-1) by murine thymocyte proliferation assay, prostaglandin E2 (PGE2) by radioimmunoassay, lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase and beta-glucuronidase using fluorometry, and collagen and casein degrading activity using radioactive substrates. Human adherent peripheral blood cells showed a proliferative response to PHA that coincided with cell maturation; BC did not inhibit PHA-induced cell proliferation of either adherent or nonadherent blood cells, indicating the non-toxic nature of these particles at the concentrations tested. BC stimulated increased release of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase; the levels of PGE2, IL-1, collagenase, and caseinase were unchanged.
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PMID:The effects of bone cement powder on human adherent monocytes/macrophages in vitro. 840 16


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