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)

Retinal pigment epithelium plasma membranes have been isolated by differential and density gradient centrifugation of glass-bead-bound, collagenase-treated cells. Electron microscopic evidence indicates that the glass-bead-bound cells were devoid of red blood cells, rod outer segments and other ocular cell contaminants. The plasma membranes were recovered in 4-6 micrograms/eye yields and purified 10-fold by 5'-nucleotidase and alkaline phosphodiesterase I, and 6.5-fold by (Na+ + K+)-ATPase. Plasma membrane purity as measured by covalent labeling of the epithelial cell plasma membrane proteins with p-(diazonium) benzene[32S]sulfonic acid was 8-19-fold. In purified plasma membranes contamination by mitochondria was undetectable and lysosomal contamination reduced 100-fold, while endoplasmic reticulum was 2-fold enriched. SDS-polyacrylamide gel electrophoresis of the plasma membrane proteins revealed 23-26 major bands by Coomassie blue staining and 12-16 major bands by radioactive labeling. The plasma membranes exhibited a 3-fold lower concentration of docosahexaenoic acid, a 3-fold higher cholesterol/phosphate ratio, and were 10-fold enriched in cholesterol per micrograms protein when compared to the whole cell fraction. Retinal epithelial plasma membranes contain an average of 1 mol cholesterol per mol of lipid phosphorus, a high palmitic acid concentration (39 mol%) and a low concentration of docosahexaenoic acid (2 mol%). The lipid profile of the retinal pigment epithelial plasma membranes indicates that they are typical of plasma membranes from many other cell types and that they appear to be less fluid than total rod outer segment membranes.
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PMID:Isolation of plasma membranes from the bovine retinal pigment epithelium. 298 2

IL 1 is a major immunoregulatory molecule produced by macrophages, and it appears to be the molecular orchestrator of nonspecific host defense mechanisms against a variety of environmental insults. Many investigators have used artificial agents to stimulate macrophages to produce IL 1. We now report production of large quantities of IL 1 after a physiologic stimulus. The Lyme disease spirochete, recently isolated and adapted for growth in vitro, was used to stimulate P388D1 cells or human peripheral blood monocytes. Spirochetes were added to confluent macrophage cultures in serum-free RPMI at a ratio of 10:1. The release of IL 1 was dose-dependent. The 24-hr supernatant IL 1 activity was determined by using the thymocyte Con A co-mitogenesis assay. Activity was not due to an endotoxin on, or produced by, the spirochete. A polymyxin B affinity column failed to remove activity, and polymyxin B in the spirochete-macrophage culture had no effect on IL 1 production. Supernatants were collected, were concentrated, and were subjected to size exclusion HPLC. Three areas of activity were found in P388D1 cell supernatants (Mr greater than 60,000, 40,000, and 20,000), whereas two peaks (Mr 23,000 and 13,000) were found in human monocyte supernatants. The Mr 20,000 and 13,000 peaks from murine and human cell supernatants, respectively, were subjected to SDS-PAGE and were shown to be single bands (Mr 12,400 for the mouse IL 1 and Mr 13,500 for the human IL 1). Isoelectric focusing of column-purified IL 1 preparations showed two different pI in both human (pI 7.25 and 4.4 to 5) and murine (pI 7.25 and 5.55) IL 1. Fibroblasts cultured with murine or human IL 1 preparations demonstrated both an increase in secreted collagenase and increased cell proliferation. Thus, a physiologic stimulus and simple biochemical techniques produce large amounts of very pure mouse or human IL 1. That this IL 1 is produced by Lyme disease spirochete-stimulated macrophages may explain some of the clinical manifestations of Lyme disease.
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PMID:Lyme disease spirochetes induce human and murine interleukin 1 production. 298 84

The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.
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PMID:Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis. 299 65

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.
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PMID:Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen. 299 41

In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis.
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PMID:Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1. 299 29

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.
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PMID:Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid. 300 Mar 52

The composition of proteins and antigens of female Onchocerca volvulus from one focus of transmission was studied by SDS-PAGE and immunoblotting. Worms that had been exposed to collagenase digestion of onchocercomata for different periods of time and parasites of different age and status of reproduction were tested. Some O. volvulus antigens were found to be sensible to prolonged digestion (molecular weights: 18 KD, 21 KD, 24 KD) but the majority of the antigens was stable up to 64 hours of incubation at 34 degrees C. The composition of proteins and antigens varied with the age and the status of reproduction of the worms. Slight differences between individual filariae were found, even when worms of comparable age and status of reproduction were tested that had been exposed to nodule digestion for comparable time.
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PMID:Studies on the protein and antigen composition of individual female Onchocerca volvulus after collagenase digestion. 300 80

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.
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PMID:Characterization of tubular basement membrane antigens in human kidney. 300 98

Gold sodium thiomalate, a drug used widely in the therapy of rheumatoid arthritis, was found to be an activator of latent human polymorphonuclear leukocyte collagenase. The activation was demonstrated by two distinct and independent collagenase assays: by recording with a spectrophotometer at 227 nm the enzyme-induced increase in ultraviolet difference absorbance of native type I collagen connected to the cleavage of collagen at 37 degrees C [(1986) Eur. J. Biochem. 156, 1-4] and by SDS-polyacrylamide gel electrophoresis analysis of formation of specific products of collagen resulting from collagenase cleavage at 25 degrees C. Activation of latent collagenase by gold sodium thiomalate appeared to be of the same magnitude as by the known activator phenylmercuric chloride.
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PMID:Gold sodium thiomalate activates latent human leukocyte collagenase. 302 35

A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.
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PMID:Purification and characterization of a streptomycete collagenase. 302 69


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