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

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.
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PMID:Activation of fibroblast procollagenase by mast cell proteases. 5 9

Previous studies have shown that there is microscopic and biochemical evidence that rat parietal yolk sac synthesizes basement membrane (type IV) collagen; this study shows that a radioimmunoassay may be used for the detection of type IV collagen in such biosynthetic systems. Rat parietal yolk sacs incubated in medium containing (14C) proline either with or without alphaalpha-dipyridyl produced either unhydroxylated or hydroxylated (14C)collagen. The immunological reactivity of these two preparations was investigated using antibodies to bovine type IV collagen in a radioimmunoassay which demonstrated that the hydroxylated (14C)collagen preparation had a considerably higher level of antigenicity than the unhydroxylated (14C)collagen. Hydroxylated rat type IV (14C)collagen which had been reduced and alkylated was intermediate in antigenicity between hydroxylated and unhydroxylated material. These findings suggest that there are antigenic determinants which depend upon hydroxylation of the collagen molecule, and others dependent upon intact disulphide bonds. In addition, various levels of pepsin extracted unlabelled calf anterior lens capsule collagen caused inhibition of antibody binding to (14C)collagen. Rat type IV (14C)collagen which had been digested with collagenase was inactive in the radioimmunoassay, while pepsin digestion caused no reduction in antigenicity. These findings suggest that the antiserum is directed towards the collagenous part of the molecule and may be a useful tool in the detection of biosynthesized basement membrane collagen.
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PMID:Rat parietal yolk sac basement membrane. An investigation of the antigenic determinants using a radioimmunoassay. 6 39

The deposition of alpha-1-antitrypsin and alpha-2-macroglobulin, both known to be inhibitors of human skin collagenase, is significantly increased in keloids and in hypertrophic scars (as compared to normal skin). However, following intralesional triamcinolone treatment, a marked resorption of these abnormal scars occurs along with a significant reduction of the alpha-1-antitrypsin deposits. These findings suggest that alpha-globulins are involved in abnormal scar formation, and that triamcinolone may remove collagenase and/or protease inhibitors--thereby allowing activation of the collagenase with subsequent breakdown and resorption of the excessive collagen.
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PMID:Tissue alpha-globulins in keloid formation. 6 77

To elucidate the mechanism of synovial damage in rheumatoid arthritis, we studied the activation of latent collagenases released from adherent rheumatoid synovial cells in culture. Latent enzyme was not complexed with alpha2 macroglobulin, the prinicpal proteinase inhibitor in serum, and could be activated by trypsin in the presence of alpha2 macroglobulin if sufficient proteinase was added to saturate inhibitor. Latent collagenase bound half as effectively to collagen fibrils as active enzyme. Plasmin was a threefold better activator of latent enzyme than trypsin and could be generated by addition of plasminogen to synovial-cell cultures. Production of both collagenase and plasminogen activator was inhibited by dexamethasone (10(-9) M). These studies emphasize in importance of control of activation in regulation collagenase activity, It is likely that rheumatoid synovium produces both latent collagenase and plasminogen activator; plasmin is activated from its zymogen, plasminogen, present in inflamed tissues, and in turn activates collagenase.
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PMID:Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator. 6 27

A cross-linked fragment (peptide T1X) with a molecular weight of 13,000 could be isolated from a tryptic digest of insoluble type III collagen of calf skin. Peptide T1X was conjugated on to bovine serum albumin by glutaraldehyde and used for immunization of rabbits. The antisera reacted in passive haemagglutination and radioimmune assay with peptide T1X, type III collagen and its constituent alpha1(III) chain. Little or no reaction was observed with type I collagen and alpha1(I) chain. While rabbit antisera to neutral salt-soluble type III Collagen also showed a strong binding for 125I-labelled peptide T1X much less reaction was observed with antisera to type I collagen. The antigenicity of type III collagen was largely destroyed by pepsin treatment suggesting that it resided in non-helical segments. A fragment of peptide T1X produced by digestion with collagenase retained antigenic activity. The data indicated that the aminoterminal region of type III collagen contains strong antigenic determinants located in a non-helical sequence of about sixteen amino acids. Antibodies to these antigenic determinants were purified and rendered specific for type III collagen by immunoadsorption. The antibodies stained in indirect immunofluorescence tests particularly those regions in various connective tissues which are rich in reticulin fibres. Different staining patterns were observed with antibodies to type I collagen.
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PMID:Production and specificity of antibodies against the aminoterminal region in type III collagen. 6 19

Specific collagenases responsible for the initial enzymic step leading to degradation of the collagen fibrils of connective tissues have been found in both latent and active forms. The most important factor controlling the local activity of collagenase extracellularly may be an inhibitor that is synthesised by connective tissues, and it is proposed that latent enzymes are all enzyme-inhibitor complexes.
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PMID:A new factor that may control collagen resorption. 6 39

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.
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PMID:Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue. 7 61

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.
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PMID:Binding of latent rheumatoid synovial collagenase to collagen fibrils. 7 45

To obtain viable cells from normal human skin, clostridial collagenase was used. Crude collagenase digestion of collagen fibres and basal lamina results in free dermal cells and sheets of epidermis. The collagenase was tested at various concentrations, solvents and incubation periods. The specimens digested were either split or full thickness skin of varying size. The optimal result was obtained by using small (3mm across) split skin pieces incubated in 2 mg/ml collagenase. The choice of solvent MEM, MEM supplemented with serum, and Tris buffer, was less important. 3 hours' incubation the epidermis was peeled off in sheets and finally dissociated by trypsin-EDTA. The corium was completely digested after 6 hours. After 6 hours' incubation no viable cells could be seen. The epidermal cells appeared mainly as polygonal cells of various sizes and a few little dendritic cells. The dermal cells had a heterogeneous morphology during the first weeks of cultivation. After 2 weeks the cells appeared as fibroblast-like cells.
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PMID:Enzymatic liberation of viable cells of human skin. 7 32


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