Purification and properties of a specific collagenase from rabbit synovial fibroblasts.
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ABSTRACT:
SUBMITTER: Werb Z
PROVIDER: S-EPMC1172413 | biostudies-other | 1975 Dec
REPOSITORIES: biostudies-other
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Project description:1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.
| S-EPMC1161646 | biostudies-other
Project description:1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.
| S-EPMC1166126 | biostudies-other
Project description:1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.
| S-EPMC1162004 | biostudies-other
Project description:Cartilage, bone and the interstitial stroma, composed largely of the interstitial collagens, types I, II and III, are remodelled by three members of the metalloproteinase (MMP) family, collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13). MMP-1 and MMP-13 may contribute directly to disease progression, since they are induced in patients with rheumatoid arthritis and osteoarthritis. The study of MMP-1 and MMP-13 gene regulation in models of arthritic disease has been problematic because mice and rats, which are typically used, only possess a homologue of MMP-13. Here we show that in contrast with mice and rats, rabbits possess distinct genes homologous to human MMP-1 and MMP-13. Furthermore, rabbit MMP-13 is expressed simultaneously with MMP-1 in chondrocytes and synovial fibroblasts in response to the cytokines interleukin-1 and tumour necrosis factor-alpha, or the phorbol ester PMA. The time course of MMP-13 induction is more rapid and transient than that of MMP-1, suggesting that distinct mechanisms regulate the expression of these two collagenases. We have cloned the rabbit MMP-13 gene from synovial fibroblasts and demonstrated that the rabbit gene shares greater homology with human MMP-13 than does the mouse interstitial collagenase. Together with the fact that mice and rats do not possess a homologue to human MMP-1, our data suggest that the rabbit provides an appropriate model for studying the roles of interstitial collagenases in connective-tissue diseases, such as rheumatoid arthritis and osteoarthritis.
| S-EPMC1219357 | biostudies-other
Project description:1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.
| S-EPMC1162866 | biostudies-other
Project description:A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.
| S-EPMC1172448 | biostudies-other
Project description:A number of human diseases, such as arthritis and atherosclerosis, include characteristic pathology in specific anatomical locations. Here we show transcriptomic differences in synovial fibroblasts from different joint locations and that HOX gene signatures reflect the joint-specific origins of mouse and human synovial fibroblasts and synovial tissues. Alongside DNA methylation and histone modifications, bromodomain and extra-terminal reader proteins regulate joint-specific HOX gene expression. Anatomical transcriptional diversity translates into joint-specific synovial fibroblast phenotypes with distinct adhesive, proliferative, chemotactic and matrix-degrading characteristics and differential responsiveness to TNF, creating a unique microenvironment in each joint. These findings indicate that local stroma might control positional disease patterns not only in arthritis but in any disease with a prominent stromal component.
| S-EPMC5376654 | biostudies-literature
Project description:We investigated the effect of heat shock on the expression of the collagenase gene in normal human synovial and dermal fibroblasts. Heat shock (42-44 degrees C for 1 h) caused a marked increase in heat-shock protein 70 (HSP-70) mRNA levels, followed by a delayed increase in collagenase mRNA levels, in both cell types. Pretreatment with cycloheximide had no effect on the heat-shock-induced increase in HSP-70 mRNA expression, but abrogated the induction of collagenase mRNA during the recovery. To study the mechanisms of collagenase-gene induction by heat shock, the transcriptional activity of a collagenase-promoter-driven chloramphenicol acetyltransferase (CAT) reporter gene was examined in transient transfection experiments. Heat shock was followed by a > 2-fold increase in CAT activity driven by a 3.8 kb fragment of the collagenase promoter, or by a construct containing an AP-1 binding site. A mutation in the AP-1 binding site abolished the effect of heat shock. Electrophoretic-mobility-shift assays revealed a marked increase in DNA-binding activity specific for the AP-1 binding site in nuclear extracts prepared from synovial fibroblasts recovering from heat shock. These results indicate that heat shock causes a delayed increase in collagenase-gene expression in human fibroblasts, and suggests that this stimulation involves, at least in part, transcriptional activation through an AP-1 binding site. Heat shock appears to initiate a programme of cellular events resulting in collagenase-gene expression, and therefore may contribute to connective-tissue degradation in disease states.
| S-EPMC1136787 | biostudies-other
Project description:Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex®--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.
| S-EPMC3286458 | biostudies-literature
Project description:1. Antisera were raised against the collagenase from rabbit synovial fibroblasts and characterized by immunoprecipitation and immunoinhibition reactions. 2. Immunoglobulins from the antisera were potent inhibitors of the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. 3. The antibody-binding fragment, Fab', produced by digesting the IgG (immunoglobulin G) with pepsin, inhibited collagenase activity just as well as whole IgG. 4. A specific antiserum to the rabbit collagenase was raised by a multi-step procedure. An initial antiserum was made by injecting partially purified collagenase as a complex with sheep alpha2-macroglobulin into a sheep. The non-specific antiserum so obtained was used to produce precipitin lines with the purified enzyme, and these lines were used as antigen for the production of the specific antiserum. 5. An IgG preparation from the specific antiserum was a specific and potent inhibitor of the rabbit synovial fibroblast collagenase. Neutral metallo-proteinase activity secreted by the rabbit fibroblasts was not inhibited by the antibody to the rabbit collagenase. 6. Criteria for determination of the specificity of antisera are discussed.
| S-EPMC1172414 | biostudies-other