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)

Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers.
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PMID:Synthesis of basement membrane collagen by cultured human endothelial cells. 5 57

A method for primary culture of ovine myometrial cells is described. After dissection, myometrium of ewe uteri was digested in trypsin and collagenase. The cells were preplated for 1 h at 37 degrees C. The non-attached cells were grown in appropriate medium supplemented with 2% fetal calf serum. They had a doubling time of 3 days, reached confluency at 10 days and did not exhibit contact inhibition. Cultures were maintained up to 22 days. Characterization of the cells was achieved by electron microscopy, analysis of myosin in cell extracts and assessment of hormone sensitivity. The cells were found to contain myofilaments, characteristic of smooth muscle. A high content of myosin (6--13%) was demonstrated on SDS-polyacrylamide gel electrophoresis: this was confirmed by ATPase activity assay. Cells responded to estradiol stimulation by increased protein synthesis, and bound [3H]estradiol in a specific and saturable way. These results suggest that myometrial cells grown in primary culture should provide a useful model for studying the hormonal control of contractile protein synthesis.
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PMID:Myometrial cells in primary culture: characterization and hormonal profile. 15 21

Explants from rabbit aortic media were incubated in MEM medium supplemented with 14C-lysine and with 10 p. 100 hyperlipemic (type IV and V) or normal human serum respectively. The incubated fragments were extracted at increasing ionic strength. The insoluble collagen and elastin were hydrolysed with collagenase and alcoholic potassium hydroxyde respectively. The radioactivity was determined in the extracts and the radioactive labelling profile of proteins was investigated on polyacrylamide gel electrophoresis in SDS. With the exception of the collagenase extract (polymeric collagen) the incorporation of the radioactivity into insoluble collagen is not altered or increases. These the incubation was carried out in the presence of hyperlipemic serum. Incorporation of the radioactivity into insoluble collagen seems not to be altered. These results show a decreased protein synthesis with a relative increase in the biosynthesis of polymeric insoluble collagen in the aortic media incubated in the presence of hyperlipemic serum.
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PMID:Action of human hyperlipemic sera on the biosynthesis of intercellular matrix macromolecules in aorta organ cultures. 18 73

The uptake of latex by fibroblasts in confluent primary culture results in the secretion of collagenase at a linear rate for a prolonged period. Phagocytosis might therefore constitute an important level of collagenase regulation in corneal ulceration. The collagenase in cell cultures is present in a latent form (40,000 MW) like that obtained from organ cultures of ulcerating corneas and can be activated proteolytically. Production of the latent collagenase in cell culture depends upon the presence of serum and diminishes greatly when serum is removed from the medium. Collagenase activity can be demonstrated after the latent collagenase has been separated from serum antiproteases in the media. Alternatively, careful titration of the crude media with trypsin to saturate serum antiproteases, to release collagenase from the complex with alpha 2-macroglobulin, and to activate latent collagenase also results in measurable collagenase activity. The collagenase that is secreted cleaves fibrillar type I collagen and cleaves soluble type I collagen into the typical 3/4 and 1/4 length fragments, as demonstrated by SDS-gel electrophoresis and electron microscopy.
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PMID:Collagenase from corneal cell cultures and its modulation by phagocytosis. 22 35

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

The extraction of isolated vertebrate smooth muscle cells at high and low ionic strength yields cell ghosts which are seen in the electron microscope to be composed of a complex network of 10-nm filaments, together with residual actin. After SDS-gel electrophoresis of the cell ghosts only 2 bands may be recognized, one corresponding to actin and the other migrating at about 55 000 mol. wt that arises from the 10-nm filaments. The 10-nm filaments are extremely sensitive to proteolysis and are absent from cells exposed to crude collagenase in the presence of Triton X-100. Such cells, lacking 10-nm filaments, still contract in response to ATP. The data indicate that the 10-nm filaments are not essential for contraction, but rather form a specialized intracellular cytoskeleton. While completely insoluble in concentrated salt solutions the 55 000 mol. wt protein is readily extracted with acetic acid from homogenized and salt-extracted smooth muscle residue. The extracted protein reassembles, on dialysis, into filaments of about 10-nm diameter and has an amino acid composition almost identical to that deduced for vertebrate neurofilaments. From the cytoskeletal role that the 10-nm filaments play in smooth muscle and, as appears likely, in other cell types the filament protein has been tentatively termed 'skeletin'. Results relating to the proportion of skeletin in smooth muscle and the structure of the 10-nm filaments are described and discussed.
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PMID:Studies on the function and composition of the 10-NM(100-A) filaments of vertebrate smooth muscle. 56 Oct 84

To elucidate the mechanisms for the presence of immunoglobulins and human serum albumin (HSA) in articular cartilage from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), the recovery of these molecules was determined in several elution steps. These steps included serial elutions with a neutral buffer to extract entrapped molecules, elution with 6 M guanidine hydrochloride to extract molecules bound by noncovalent interactions, and digestion of cartilage with bacterial collagenase to release molecules covalently bound to cartilage matrix proteins. Significantly more IgG than HSA was recovered with 6 M guanidine after serial elutions with neutral buffer from the cartilages of patients with both RA and OA, consistent with the binding of IgG by antigen-antibody bonds. Degradation of cartilage with collagenase released additional IgG and HSA. Analysis of the IgG and HSA, recovered with guanidine or with collagenase, using SDS-PAGE and transfer blotting, indicated for the first time the presence of disulfide bonds between these molecules and cartilage matrix molecules.
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PMID:IgG is bound by antigen-antibody bonds and some IgG and albumin are bound by intermolecular disulfide bonds to cartilage in rheumatoid arthritis and osteoarthritis. 131 84

Collagen degradation is thought to be an integral part of the healing sequence of intestinal anastomoses, but almost nothing is known about the enzyme activities involved. We have studied collagenolytic activities, extracted from 1 day-old intestinal anastomoses in the rat. Using either soluble type I collagen or fibrillar type I or type III collagen as a substrate, activities measured in extracts from anastomotic segments were compared to those in extracts from uninjured intestine, removed at operation: in all cases, the collagenolytic activity in anastomotic extracts was significantly higher. This increase was significantly more pronounced in large bowel than in small bowel. The activities were strongly inhibited by serum and metallo-chelating compounds. Analysis, by means of SDS-polyacrylamide gel electrophoresis, of the reaction products of the degradation of fibrillar type I collagen by the extracts revealed the presence of a multitude of fragments, amongst them TcA fragments characteristic for the activity of mammalian collagenase. Thus, the degradative capacity towards various collagen substrates is enhanced in the anastomotic area during the first postoperative period and a true mammalian collagenase is one of the enzymes present.
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PMID:Collagenolytic activity in experimental intestinal anastomoses. Differences between small and large bowel and evidence for the presence of collagenase. 131 43

Conglutinin and mannose-binding protein (MBP) are members of the C-type lectins which are widely present in mammalian plasma. Serum amyloid P-component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tetramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc-like molecules (diameter 11 nm) in the SAP preparations.
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PMID:Purification, subunit characterization and ultrastructure of three soluble bovine lectins: conglutinin, mannose-binding protein and the pentraxin serum amyloid P-component. 131 9

A method specific for identification of collagens irrespective of type, species, or tissue origin, and of their derived fragments of molecular weight more than 10,000, is described. The method is based on the low-temperature affinity between clostridial collagenase and almost all types of collagens as well as on the affinity between collagenase and its antibodies. Various collagens or fragments derived from them by treatment with CNBr were separated by SDS-PAGE and immobilized onto a nitrocellulose membrane by a slot-blot technique or electrotransfer. Following binding of clostridial collagenase to a collagen or its fragments at 0 degrees C, the collagen-collagenase complex was fixed with glutaraldehyde. The complex was then allowed to bind anti-collagenase antibody at room temperature. The new complex was subsequently treated with 125I-labeled donkey anti-rabbit IgG and visualized as an autoradiogram. Under the conditions of low temperature used, the collagenase binds to collagens without causing their digestion. This procedure is specific for detection of soluble collagens as well as of insoluble collagens converted to fragments by treatment with CNBr. The method is uniquely suited for detection of fragments of tissue collagens. Also, it may serve as a prototype for methods for detection of other specific polymeric substances.
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PMID:Specific identification of collagens and their fragments by clostridial and anti-collagenase antibody. 132 68


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