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)

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) plays an important role in extracellular matrix turnover and thereby modulates atherosclerotic plaque development. MMP-1, -2, -3, and -9 activity is increased by atherosclerosis, but the status of TIMPs is less clear. We therefore compared secretion of TIMPs-1 and -2 from cultured aortic explants derived from arch, middle, and distal portions of thoracic aortas of normal rabbits and rabbits fed a 1% cholesterol diet for 8 weeks, using reverse zymography of conditioned media. Cholesterol feeding significantly increased secretion of TIMP-1 from arch and middle portions (both 2.6-fold), accompanied by 2.0- and 2.7-fold increases in TIMP-2, respectively. Atherosclerotic aortas exhibited increased immunoreactive TIMP-1 and TIMP-2 in endothelial cells, smooth muscle cells, and macrophages. Staining of extracellular matrix was also prominent within the noncellular boundary region between fibrous cap and the lipid core, and within the lipid core. Increased TIMP-2 staining was also found in the media subjacent to the lipid core. In situ gelatin zymography demonstrated excess MMP activity within the plaque with partial inhibition in the lipid core base and subjacent media, consistent with the distribution of TIMPs. Casein zymography and in situ zymography demonstrated that increased caseinolytic activity was confined to the pericellular zones of macrophages within the lipid core, again consistent with its restriction by TIMPs. In summary, atherosclerosis increases TIMP expression, which counterbalances, in part, increased MMP activity.
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PMID:Increased secretion of tissue inhibitors of metalloproteinases 1 and 2 from the aortas of cholesterol fed rabbits partially counterbalances increased metalloproteinase activity. 1039 88

A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.
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PMID:Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2. 1070 65

Enzymes currently used to tenderize meat are not substrate-specific, resulting in extensive myofibrillar protein degradation that often produces an undesirable texture. Bovine placental metalloproteases, which selectively hydrolyze connective tissue proteins while leaving myofibrillar proteins intact, may tenderize meat without causing texture problems. Therefore, our objective was to extract and crudely purify bovine metalloproteases from bovine placenta for possible use as tenderizers in meat systems. Enzymes were extracted from homogenized tissue and purified by ammonium sulfate precipitation. Samples were collected before (crude enzyme) and after gel filtration on a Sephadex G-100 column. Spectrophotometric analysis identified one major peak (filtered enzyme). Gelatin, casein, and type I acid-soluble collagen zymography were used to determine substrate specificity. Beef myofibrillar proteins were incubated with crude and filtered enzyme fractions, enzymes quenched, and substrate degradation visualized using SDS-PAGE. Active gelatinases and collagenases exhibiting molecular weights of 57 to 65 kDa were detected on zymograms. Banding patterns from crude enzyme indicated two enzymes with both gelatinase and collagenase activity and a third enzyme with gelatinase activity only. Banding patterns from filtered enzyme indicated two enzymes with both gelatinase and collagenase activity. Proteolytic activity was not detected with casein, actin, or myosin heavy-chain substrates. Due to specificity for collagen and gelatin, these enzymes may be capable of improving the tenderness of certain cuts relatively high in connective tissue, while avoiding myofibrillar protein hydrolysis.
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PMID:Bovine placental protease specificity toward muscle connective tissue proteins. 1090 28

We investigated cells and conditioned media of the three human keratinocyte cell lines HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic) with regard to production and secretion of the collagenases-1 to -3 (MMP-1, MMP-8 and MMP-13) and TIMP-1 using semi-nested RT-PCR, Western blots, ELISA, immunocytochemistry and casein zymography. Transcripts of MMP-1, -8, -13 and TIMP-1 were detected in all cell lines by RT-PCR and the corresponding proteins were found in the cytoplasm of all three cell lines by Western blot analysis and/or immunocytochemistry. The conditioned media of the malignant II-4RT cells contain significantly more MMP-1 and MMP-8 than those of HaCaT or A5 as evidenced by immunoblotting and ELISA. In addition to the presence of latent MMP-1, zymography also detected the active form of this enzyme. TIMP-1 was found only in extracts of all three cell lines, predominantly in A5. This study clearly indicates that the epithelial tumor cells synthesize different collagenases and TIMP-1. The malignant clone secretes increased amounts of distinct collagenases compared to the non-tumorigenic cell line, thereby verifying a correlation between biological behaviour and the amount of collagenases. In addition, we provide clear evidence that MMP-8 is not exclusively found in polymorphonuclear granulocytes, but also in keratinocyte cell lines.
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PMID:Human keratinocyte cell lines differ in the expression of the collagenolytic matrix metalloproteinases-1,-8, and -13 and of TIMP-1. 1093 83

In urodele amphibian spinal cord regeneration, the ependymal cells lining the central canal remodel the lesion site to favor axonal regrowth. We profiled the production of matrix metalloproteinases by injury-reactive mesenchymal ependymal cells in vivo and in vitro and found that matrix metalloproteinases are involved in this remodeling process in the axolotl (Ambystoma mexicanum). The production of cell-associated matrix metalloproteinases in vivo was shown to be identical to that in our cultured ependymal cell model system. Activated and zymogen forms of matrix metalloproteinases were identified using zymography, chemical inhibitors of matrix metalloproteinases, and cleavage of propeptides by organomercurials. The principal cellular proteinases consisted of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials. In addition, ependymal cell conditioned medium contained secreted forms of the enzyme undetectable in situ. Matrix metalloproteinase-9 (gelatinase B) as well as matrix metalloproteinase-2 and matrix metalloproteinase-1 were secreted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase-3 (prostromelysin) secreted into the culture medium. Matrix metalloproteinases were still present at 4 weeks post-lesioning when the ependymal cells have just re-epithelialized, but decreased near the completion of regeneration (8 weeks post-lesioning). Zymography showed no detectable matrix metalloproteinases in unlesioned cord but the presence of tissue inhibitor of metalloproteinase-1 in intact cord was seen by Western blotting. This study shows that matrix metalloproteinases are associated with urodele spinal cord regeneration and validates the use of our ependymal cell tissue culture model system to evaluate ependymal cell behavior during spinal cord regeneration.
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PMID:Matrix metalloproteinase production in regenerating axolotl spinal cord. 1101 20

Rats were studied to determine if matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were detectable in developing embryonic and uterine tissues during the implantation period; that is, Days 6-8 of pregnancy. Tissue extracts were studied by zymography, reverse zymography and Western blotting. Immunohistochemistry procedures using antibodies against MMPs and TIMPs were applied to tissue sections that passed through implantation sites. The MMP inhibitor, doxycycline, was injected intraluminally into uterine horns of pseudopregnant animals. MMP-2 and -9, and TIMP-2 were detected in gelatin and reverse zymographs, respectively, from extracts of implantation sites on each day studied. Western blots defined bands corresponding to MMP-1, but casein zymographs did not consistently show evidence of extractable MMP-1 or -3. In tissue sections the primary decidual tissue area stained consistently for TIMP-2 and on Days 7 and 8 this area also had positive staining for MMP-1 in close proximity to the implanting embryo. On Day 8 positive staining in the outer stromal tissue was detected using an antibody against MMP-9, and another MMP (possibly MMP-3) was localized exclusively in ectoplacental cone/trophoblastic cells. Intraluminal injection of doxycycline did not significantly prevent the growth of the uterine horns following surgical induction of pseudopregnancy on Day 5. The strong localization of TIMP-2 in the primary decidual tissue is similar to that reported previously for TIMP-3, indicating that these inhibitors have a role in decidualization, including the regulation of trophoblastic invasion.
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PMID:Matrix metalloproteinases and their endogenous inhibitors during the implantation period in the rat uterus. 1110 Dec 74

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.
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PMID:Differential regulation of metalloproteinase production, proliferation and chemotaxis of human lung fibroblasts by PDGF, interleukin-1beta and TNF-alpha. 1113 72

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.
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PMID:Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes. 1133 58

Unlike the gelatinases (MMP-2 and -9), matrilysin (MMP-7) and collagenases (MMP-1 and -13) are difficult to detect at low levels in conventional casein or gelatin zymography. In this report, heparin was used to enhance the zymographic assays for MMP-7, -1, and -13. With the addition of heparin to the enzyme sample, MMP-7 can be detected at a level of 30 pg in transferrin zymography and MMP-1 and -13 can be detected at a level of 0.2 ng in gelatin zymography. Carboxymethylated transferrin is used instead of casein as a substrate for assaying rat MMP-7. This substrate does not require a prerun step or substrate cross-linking to give uniform staining and clear band formation. It is necessary for heparin to run to the same region of the gel as the enzyme to produce its enhancing effect. For MMP-7 movement of heparin and enzyme is almost equal; for the collagenases it is necessary to add heparin to each well after the electrophoretic run is underway. Possible mechanisms of activity enhancement are discussed.
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PMID:Heparin-enhanced zymographic detection of matrilysin and collagenases. 1137 76

Atherogenesis requires extracellular matrix (ECM) alterations, a process possibly mediated by matrix-degrading metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). The objective of this study was to examine the immunohistochemical expression patterns of MMPs-1, -2, -3 and -9 and their tissue inhibitors, TIMPs-1, -2, -3 and -4 during the three major stages of atherosclerotic lesion development in hypercholesterolemic Syrian Golden hamsters. Aortic atherosclerotic lesions (fatty streak, fibro-fatty and advanced) were histologically characterized in treated hamsters at 12, 24, and 49 weeks. The immunochemistry expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. MMP activity in control aortas and atherosclerotic lesions was characterized by in-situ zymography. Positive immunoreactivity for MMPs-2, -3, -9 and TIMPs-1, -2,-3, and -4 was observed in both control and atherosclerotic aortic arch segments, while MMP-1 was only observed in atherosclerotic lesions. Using in-situ zymography, we identified casein and gelatin degradation in fatty streak, fibro-fatty and advanced lesions. The immunohistochemical expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. In all lesion stages, substrate degradation was inhibited with 1,10-phenanthroline. Degradation of these substrates was not observed in control aortas. In addition, substrate degradation was inhibited with 1,10-phenanthroline. These findings suggested that in control segments, the net proteolytic balance was shifted in favor of MMP inhibition. Alternatively, despite the colocalization of MMPs and TIMPs in the treated segments, net proteolytic balance favored the catalytic MMPs.
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PMID:Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: correlation with in-situ zymography. 1184 55


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