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)

A preparation rich in basement membranes isolated from rat testes (STBM) was exposed to pepsin, collagenase, trypsin, and pronase to obtain soluble fractions. The immunological reactivity of these fractions was studied by gel immunodiffusion or by passive hemagglutination tests against an anti-STBM serum. All fractions reacted with the antiserum, but the highest titer was detected when the antiserum was reacted with a fraction that contained only traces of hydroxyproline (fraction 1), whereas low titers were obtained with collagen or collagen fragments isolated from STBM. Antibodies in the anti-STBM serum were mainly directed to the glycoproteins of STBM not related to collagen. Fraction 1, obtained by subsequent collagenase and trypsin digestion of STBM and purification by Sephadex G-200, was a high molecular weight glycoprotein that was free of half-cystine and methionine, had only traces of hydroxyproline, and contained 7.2% neutral sugars, 0.26% sialic acid, and 8.7 residues of glucosamine per 1000 residues of amino acids.
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PMID:Isolation and immunological reactivity of soluble fractions from rat seminiferous tubule basement membrane. 9 Apr 90

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.
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PMID:Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix. 36 26

Dispersed cell preparations enriched in beta-cells were obtained by collagenase digestion of fetal bovine pancreas and separation by Ficoll gradient centrifugation. These cells actively incorporated [3H]leucine into proinsulin and insulin. Incubation of these cells in the presence of the arginine analogue, L-canavanine, resulted in the inhibition of conversion of newly formed proinsulin to insulin and the appearance of a radioactive component of molecular weight 11,000-12,000. Incorporation of [35S]methionine into this component was detected in the presence of canavanine, an event not observed in control incubation. Canavanine thus induced the formation of a component possessing molecular weight and compositional properties expected for preproinsulin. Further characterization of cellular products by polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate showed a highly labeled band corresponding to molecular weight 18,000-20,000 which might be involved in insulin biosynthesis.
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PMID:Preparation of beta-cells from fetal bovine pancreas: characterization of insulin biosynthetic activity. 38 31

We have recently reported that lipase may play a role in the pathogenesis of acute pancreatitis by its ability to release fatty acids from triglycerides. The aim of this study was to further investigate the effect of lipase and its various digestive products on the integrity of isolated pancreatic rat acini. Pancreatic acini were prepared by collagenase digestion and their newly synthesized proteins labeled with 35S-methionine. Acini were later incubated in buffer to which various factors were added: Products of lipolytic digestion, such as various fatty acids and monoglycerides, fat tissue, nonactivated or trypsin activated homogenized pancreatic tissue, and a specific lipase inhibitor (THL, tetrahydrolipstatin). Cellular destruction was quantified by the degree of radiolabeled proteins released. Short chain fatty acids and monoglycerides (up to C-12) caused cellular destruction, whereas long chain fatty acids and their respective monoglycerides were not harmful. With regard to unsaturated fatty acids, long chain fatty acids (C-18 to C-22) were also able to destroy cells. The degree of cellular necrosis correlated with incubation time and fatty acid concentration. The cellular damage caused by incubation of acini with either inactive or trypsin activated pancreatic homogenates together with triglycerides could be completely inhibited by the specific lipase inhibitor THL. Bile alone caused no damage. When bile was combined with activated-pancreatic homogenates, about 25% of newly synthesized proteins were released by acini within 30 min. Incubation with a combination out of bile activated pancreatic homogenates and triglycerides resulted in the most pronounced damage. This acinar destruction could only be partly inhibited by THL. These studies suggest that both lipase and phospholipase-A2 may play an important role in the pathogenesis of acinar cell destruction.
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PMID:Isolated rat pancreatic acini as a model to study the potential role of lipase in the pathogenesis of acinar cell destruction. 128 21

Activated macrophages (M phi s) have terminal alpha-D-galactosyl (alpha D-Gal) residues on their membranes that are not apparent on resting cells. Ligation of these epitopes with Griffonia simplicifolia I-B4 (GSI-B4), a lectin that has specificity for alpha-D-Gal residues, alters selected M phi functions. To explore the mechanism(s) that may be responsible for some of the functional changes, alterations in the secretory pattern of [35S]methionine-labeled proteins were assessed when cells were cultured with or without this ligand. The proteins were identified by Western blots and quantitated. Interestingly, alpha-D-Gal ligation proved to decrease the secretion of some proteins while increasing the secretion of others. Some of the most significant changes were observed in four proteins: fibronectin and transglutaminase were down-regulated by 55 and 66% respectively, while plasminogen activator inhibitor type 2 was increased by 259% and collagenase was increased 1000-fold. These observations show that the emergence of new oligosaccharide epitopes, such as alpha-D-Gal, concomitant with M phi activation may serve to mediate the transduction of signals that cause quantitative changes in the elaboration of diverse M phi products. The biologic significance of the four identified proteins has been well established. Fluctuations in their levels are likely to play a role at sites of chronic inflammation.
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PMID:Specific ligation of surface alpha-D-galactosyl epitopes markedly affects the quantity of four major proteins secreted by macrophages. 137 96

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

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.
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PMID:Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity. 144 34

A genetic approach to define the role of collagenase in physiological and pathological bone remodeling is to identify spontaneous mutations in the collagenase gene which alter enzymatic activity. Alternatively it is possible, though site-directed mutagenesis, to alter genes encoding critical amino acid sequences in the collagen substrate, in a manner analogous to the successful development of animal models for osteogenesis imperfecta. We have thus utilized this approach to alter the Col1a1 gene to encode amino acid substitutions in sequences around the known collagenase cleavage site (glycine-isoleucine at positions 775-776) in type I collagen, and transfect these genes into homozygous Mov-13 fibroblasts, in which the endogenous Col1a1 gene is inactive. Nonconservative substitutions of proline for isoleucine at the P1' site and double substitutions of proline for glutamine (P2) and alanine (P2') resulted in type I collagen resistant to hydrolysis by collagenase. Furthermore, in normal fibroblasts transfected with a mutant Col1a1 gene encoding collagenase resistance in which an additional methionine substitution at position 776 provided a marker for the mutant protein, mutant and wild type triple helical molecules were synthesized and secreted as heterotrimers. A single mutant alpha 1(I) chain did not prevent cleavage of the wild type alpha 1(I) chain but it is likely that the uncleaved alpha 1(I) chain would prevent dissociation of the triple helical fragments containing the other cleaved chains. Introduction of these genes into transgenic mice should result in abnormal phenotypes characterized by altered connective tissue remodeling.
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PMID:Site-directed mutagenesis of type I collagen: effect on susceptibility to collagenase. 148 89

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.
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PMID:Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro. 151 Jul 90

The temporal aspects and mechanisms of the regulation of the matrix metalloproteinase (MMP) 72-kDa gelatinase/type IV collagenase (MMP-2) by transforming growth factor-beta 1 (TGF-beta 1) were investigated in early passage human gingival fibroblasts and compared with the regulation of the genes for collagenase (MMP-1) and TIMP, the tissue inhibitor of MMPs. Northern hybridization analyses revealed that 1.0 ng/ml TGF-beta 1 increased the abundance of MMP-2 mRNA/cell approximately 1.5-fold at 24 h, an increase similar to that observed in the level of [35S]methionine pulse-labeled MMP-2 at 24 h (1.9-fold). At 48 and 72 h, the increase in MMP-2 mRNA abundance remained elevated by 1.5-2.2-fold on a per cell basis whereas TIMP mRNA levels were elevated by up to 3.3-fold. In contrast, the relative levels of collagenase mRNA were reduced by 66-75%. The changes in the MMP-2, collagenase, and TIMP mRNA concentrations in response to TGF-beta 1 were blocked by cycloheximide indicating that protein synthesis was required to mediate the effects of TGF-beta 1 on these mRNA levels. TGF-beta 1 was also found to increase the half-life of the MMP-2 mRNA from approximately 46 to approximately 150 h but did not alter the stability of TIMP mRNA (t1/2 approximately 60 h). Nuclear run-off transcription assays revealed that MMP-2 gene transcription was increased approximately 5-fold 7 h following TGF-beta 1-treatment but returned to control levels by 24 h. In comparison, increased TIMP gene transcription was only detectable after 24 h whereas collagenase gene transcription, although low in control cells, was undetectable at 24 h. Gene transcription, mRNA levels, and message stability of the genes for the extracellular matrix proteins type I collagen and fibronectin were also increased by TGF-beta 1. Thus, the similarity in the control of MMP-2, alpha 1 (I) procollagen, and fibronectin expression at the transcriptional and post-transcriptional levels indicates that these genes may share regulatory elements. In comparison, TGF-beta 1 reduced the level of collagenase mRNA and increased the level of TIMP mRNA as a result of altered transcriptional activities, through pathways that required protein synthesis, and without changes in mRNA stability.
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PMID:Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. 164 34


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