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)

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

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface, was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000. Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE. Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).
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PMID:Radio-iodination of plasma membranes of toad bladder epithelium. 37 44

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

The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.
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PMID:Isolation of the pericellular matrix of human fibroblast cultures. 38 22

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.
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PMID:Collagen synthesis by bovine aortic endothelial cells in culture. 39 Dec 67

The elaboration of leukocyte chemotactic factors by human fibroblasts was studied. 12 lines of normal fibroblasts obtained by skin biopsy and then cultured in vitro produced chemoattractants (assessed by modified Boyden-chamber techniques) for both peripheral blood polymorphonuclear leukocytes and monocytes (obtained by Hypaque-Ficoll and dextran sedimentation). Chemotactic activity was not present performed in fibroblasts, and cycloheximide blocked its elaboration. The chemotactic activity of crude-culture supernate was heat stable (56 degrees C for 30 min), trypsin- and pronase-sensitive, and neuraminidase resistant. Characterization of the chemotactic activity by gel filtration (Sephadex G-75) showed two active fractions, one with mol wt greater than 100,000 and the other less than 10,000. In studies designed to relate these chemotactic factors to collagen, we have confirmed that type I collagen and alpha 1-chain; are chemotactically active for monocytes but not polymorphonuclear leukocytes. However, the chemotactic activity in fibroblast-culture media was media was distinct from collagen in that it attracted neutrophils, it was not precipitated by 25% ammonium sulfate, and it was resistant to collagenase treatment; ascorbic acid, in concentrations known to stimulate fibroblast collagen synthesis, had no effect on the elaboration of the chemotactic factors. Furthermore, amino acid analysis of Sephadex G-75 fractions with chemotactic activity failed to reveal amino acids such as hydroxyproline characteristic of collagen. In addition to the chemotactic factors secreted by fibroblasts, a heat-resistant factor (30 min at 56 degrees C) which generated the chemotactically active fragment of C5 (C5a) from human serum was also secreted. The elaboration of mediators of the inflammatory and immune responses by fibroblasts may initiate and(or) modulate local skin inflammatory reactions and play a protective role in vivo.
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PMID:Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts. 43 25

Elastin was extracted from human aortic plaque and adjacent grossly normal intima by the following methods: (1) 0.1 N NaOH at 100 degrees C, (2) hot NaOH and 0.2 M EDTA, (3) 5 M guanidine--HCl and collagenase, (4) guanidine--collagenase and dithioerythritol--urea--sodium dodecyl sulfate, (5) guanidine--collagenase and EDTA, (6) 10% NaCl and collagenase, and (7) NaCl--collagenase and EDTA. All elastin samples contained small amounts of carbohydrate and hydroxyproline. The lipid content of non-plaque intimal elastin samples was small (2--3%), whereas it increased to 4--6% in plaque intima. The lipid composition of elastin preparations varied significantly with the extraction procedure. Elastin from plaque intima contained significantly more cholesterol (50--60%) and less triglyceride and phospholipid than elastin of non-plaque intima (30--50% cholesterol). The contents of free and esterified cholesterol were comparable in all preparations. The main phospholipid in all samples was sphingomyelin, which comprised between 50 and 80% of the total phospholipid. Compared with NaOH-purified elastin, the other elastin samples were characterized by an increased phosphatidyl--choline content, while they all contained an almost equal amount of phosphatidylethanolamine. In elastin samples from plaque intima, the polar amino acids were increased, whereas cross-linking amino acids were decreased. The polarity and hydroxyproline content of elastin samples were slightly decreased after treatment with EDTA or dithioerythritol--urea--sodium dodecyl sulfate.
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PMID:Elastin--lipid interaction action in the arterial wall. Part 1. Extraction of elastin from human aortic intima. 46 28

Studies on the effects of bovine aorta extracts on endothelial and smooth muscle cells cultured from the same tissue indicate they contain at least four separate molecular species capable of inhibiting cell growth. Two, heparin and a recently characterized dermatan sulfate-chondroitin sulfate proteoglycan, are large polyanions and inhibit the growth of both smooth muscle and endothelial cells, but require large amounts to induce this effect. The other two are low molecular weight molecules which inhibit only the growth of endothelial cells, one of which is a protease inhibitor which has been purified to homogeneity. The fourth and most potent is an as yet unidentified fraction distinguishable from the protease inhibitor. Heparin, as reported by others, and the proteoglycan also enhance collagenase activity. It would therefore appear that aorta contains several molecular species which have the potential to affect both invasive and proliferative processes.
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PMID:Growth regulators in connective tissues. II. Evidence for the presence of several growth inhibitors in aortic extracts. 53 30

When attempts at teasing adult Dipetalonema streptocerca free from biopsy specimens of human skin proved futile a digestion procedure was initiated. Punch biopsy specimens fixed in Michel's solution (ammonium sulfate) were incubated at 25 degrees C for 3 days in a 1.0% solution of collagenase in tris-HCl buffer. Intact worms were carefully teased out of the digested collagen and camera lucida drawings and measurements were then possible. This marks the first description of intact D. streptocerca adults recovered from man.
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PMID:Recovery of intact male and female Dipetalonema streptocerca from man. 57 Dec 13

Cells obtained from chick embryo tendons incorporate isotopically labeled glucosamine and mannose into the pro-alpha1 and pro-alpha2 chains of procollagen as judged by sodium dodecyl sulfate-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The label was further localized to the propeptides of pro-alpha1 and pro-alpha2 by its chromatographic behavior after digestion with bacterial collagenase or alpha-chymotrypsin. Carbohydrate analysis of isolated pro-alpha chains showed the presence of labeled galactosamine in addition to mannose and glucosamine. Resistance to mild alkaline hydrolysis suggested that greater than 90% of the oligosaccharide units are not linked to the propeptide backbone by either serine or threonine.
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PMID:Carbohydrate moieties of procollagen: incorporation of isotopically labeled mannose and glucosamine into propeptides of procollagen secreted by matrix-free chick embryo tendon cells. 106 Nov 25


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