Purification of rabbit bone inhibitor of collagenase.
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ABSTRACT:
SUBMITTER: Cawston TE
PROVIDER: S-EPMC1162866 | biostudies-other | 1981 Apr
REPOSITORIES: biostudies-other
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Project description:1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 X 10(-10) M. 2. The molecular weight of the enzyme-inhibitor complex was found to be 54 000, but no enzyme activity could be recovered from the complex after treatment with either mercurials or proteinases. The complex thus differed from latent collagenase in terms of size, susceptibility to mercurials and behaviour on concanavalin A-Sepharose. 3. The interaction of the purified components was compared with that of crude collagenase and crude inhibitor in culture medium. Mercurial treatment partially reversed the inhibition in the crude system, but not when the purified components were used. 4. The significance of the results is discussed in relation to the extracellular control of the activity of collagenase.
| S-EPMC1154361 | biostudies-other
Project description:Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.
| S-EPMC1183665 | 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:1. Inhibition of collagenase from rabbit bone cultures by the chelating agents 1,10-phenanthroline and EDTA is almost completely reversed by Zn2+; other metal cations are less effective in reversing the inhibition. Optimal restoration of activity is achieved at Zn2+ concentrations below that of the chelator, but excess of Zn2+ is inhibitory. 2. Prolonged incubation of collagenase with either chelator causes irreversible inactivation. This inactivation is prevented by Zn2+ at the same concentrations needed to reverse the primary inhibition. 3. Collagenase incorporates 65Zn by exchange when incubated with 1,10-phenanthroline and Zn2+ containing this radioactive isotope. The 65Zn2+ can be removed from its binding site in collagenase by 1,10-phenanthroline or EDTA. Irreversible inactivation of collagenase by chelators destroys its ability to incorporate 65Zn2+. 4. Latent collagenase, the inhibited form in which collagenase first appears in culture, behaves similarly to the active enzyme in 65Zn2+-exchange experiments, but is resistant to irreversible inactivation by chelators. 5. It is concluded that collagenase is a zinc metalloenzyme that forms an inactive and unstable apoenzyme on treatment with chelators. The bound inhibitor component of latent collagenase evidently stabilizes the apoenzyme.
| S-EPMC1162853 | biostudies-other
Project description:1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.
| S-EPMC1172413 | biostudies-other
Project description:PurposeWe evaluated the use of collagenase treatment to generate a rabbit model of keratoconus and the impact of violet light (VL) irradiation on the disease model in six Japanese White rabbits.MethodsAfter epithelial debridement, the collagenase group was treated with a collagenase type II solution for 30 min; the control group was treated with a solution without collagenase. Three rabbits also underwent VL irradiation (375 nm, irradiance 310 μW/cm2) for 3 h daily for 7 days after topical collagenase application. Slit-lamp microscopy results, steep keratometry (Ks), corneal astigmatism, central corneal thickness, and axial length were examined before and after the procedure. The corneas were obtained on day 7 for biomechanical evaluation.ResultsA significant increase in Ks and corneal astigmatism was observed in the collagenase and VL irradiation groups compared with the control group on day 7. No significant difference was found in the change in corneal thickness between the groups. The elastic modulus at 3, 5, and 10% strain was significantly lower in the collagenase group than in the control group. There was no significant difference in the elastic modulus at any level of strain between the collagenase and VL irradiation groups. The average axial length at day 7 was significantly longer in the collagenase and VL irradiation groups than in the control group. Collagenase treatment induced a model of keratoconus by steepening the keratometric and astigmatic values. There was no significant difference in the observed elastic behavior of normal and ectatic corneas under physiologically relevant stress levels.ConclusionVL irradiation did not cause regression of corneal steepening in a collagenase-induced model during short-term observation.
| S-EPMC10246739 | biostudies-literature
Project description:1. An inhibitor of collagenase of apparent mol.wt. 28000 was isolated from term human amniotic fluid. 2. It is active against mammalian collagenases from a number of species and tissues as well as other mammalian metalloproteinases, but has no activity against bacterial metalloproteinases. 3. Activity is destroyed by treatment with either trypsin or 4-aminophenylmercuric acetate, by heat, and by reduction and carboxymethylation. 4. All the properties observed suggest that it is similar to the synthesized tissue inhibitor of metalloproteinases.
| S-EPMC1162867 | biostudies-other
Project description:Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.
| S-EPMC5010206 | biostudies-literature
Project description:IntroductionGlucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling.MethodsThe effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry.ResultsThe administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.ConclusionsOur data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.
| S-EPMC3132029 | biostudies-literature
Project description:Cortical bone porosity is intimately linked with remodeling, is of growing clinical interest, and is increasingly accessible by imaging. Thus, the potential of animal models of osteoporosis (OP) to provide a platform for studying how porosity develops and responds to interventions is tremendous. To date, rabbit models of OP have largely focused on trabecular microarchitecture or bone density; some such as ovariectomy (OVX) have uncertain efficacy and cortical porosity has not been extensively reported. Our primary objective was to characterize tibial cortical porosity in rabbit-based models of OP, including OVX, glucocorticoids (GC), and OVX + GC relative to controls (SHAM). We sought to: (i) test the hypothesis that intracortical remodeling is elevated in these models; (ii) contrast cortical remodeling and porosity in these models with that induced by parathyroid hormone (1-34; PTH); and (iii) contrast trabecular morphology in the proximal tibia across all groups. Evidence that an increase in cortical porosity occurred in all groups was observed, although this was the least robust for GC. Histomorphometric measures supported the hypothesis that remodeling rate was elevated in all groups and also revealed evidence of uncoupling of bone resorption and formation in the GC and OVX + GC groups. For trabecular bone, a pattern of loss was observed for OVX, GC, and OVX + GC groups, whereas the opposite was observed for PTH. Change in trabecular number best explained these patterns. Taken together, the findings indicated rabbit models provide a viable and varied platform for the study of OP and associated changes in cortical remodeling and porosity. Intriguingly, the evidence revealed differing effects on the cortical and trabecular envelopes for the PTH model. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..
| S-EPMC7702175 | biostudies-literature