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.
1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 × 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.
Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.
1 E ects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea-induced rat mammary tumours in vivo were investigated. 2 At a dose of 0.5 mg kg 71 body weight, EB1089 caused signi®cant inhibition of tumour progression over the 28 day treatment period in the absence of a signi®cant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 mg kg 71 ) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death. 3 Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3' DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 mg kg 71 ) for 14 days. 4 E ects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF-7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089.5 The e ects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and bax were examined by Western analysis. In MCF-7 cell cultures treated with 1,25(OH) 2 D 3 or EB1089 (1610 78 M), bcl-2 protein levels were decreased in a time-dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl-2/bax ratio favouring increased susceptibility of MCF-7 cells to undergo apoptosis. 6 These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.
The binding of collagenase to both a*-macroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to az-macroglobulin although no transfer of collagenase to az-macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule.
The levels of metalloproteinases and metalloproteinase inhibitors were measured in rheumatoid synovial fluid. Reliable estimates of total enzyme and inhibitor levels in the synovial fluids were obtained only after hyaluronidase treatment and gel filtration. Three latent metalloproteinases were found which, after activation, degraded collagen, proteoglycan, and gelatin. These enzymes closely resembled the metalloproteinases secreted into connective tissue culture medium. In addition to aZ macroglobulin, an M, 30,000 collagenase inhibitor was detected which closely resembled the tissue inhibitor of metalloproteinase found in tissue culture medium.Rheumatoid arthritis is a disease in which a proliferating synovial pannus degrades cartilage matrix causing joint tissue damage. Little is known of the mechanism by which the cartilage collagen and proteoglycan are lost in vivo, although a number of proteolytic enzymes present in the tissues are capable of digesting these substrates (1). Much of our knowledge of the degradative enzymes secreted by the synovium has come from in vitro tissue and cell culture techniques.Collagenase is likely to be an important enzyme involved in these processes. This metalloproteinase cleaves all 3 cr chains of the triple helical collagen molecule at one point to give characteristic threefourth and one-fourth products. The enzyme is often tis and Rheumatism Council.form August 22, 1983. secreted in a latent form and collagenase activity often cannot be detected in the culture medium of many connective tissues unless the medium is first treated with trypsin (2,3), chaotrophic agents (4-6), or mercurials (7-9). A number of inhibitors of collagenase have been described (10). In addition lo cr2 macroglobulin and p, anticollagenase, a specific collagenase inhibitor has been detected and purified from culture medium (1 1). This inhibitor is thought to play a vital role in the extracellular control of collagenase activity. We have been interested in discovering to what extent these in vitro tissue culture results reflect the situation in vivo, and have therefore examined synovial fluid from rheumatoid joints. It is likely that if cartilage erobion is taking place, the level of either collagenase or inhibitor proteins would change and give an indication of the disease activity. Previous studies (12-18) have shown that both active and latent collagenases are pregent in some rheumatoid synovial fluid. However, no cprrelation was found between the amount of enzyme detected and the disease activity, and the amounts of collagenase inhibitors were not measured in these studies.In this report we have assayed for metalloproteinases and mctalloproteinase inhibitors in rheumatoid synovial fluids taken from patients with active disease. MATERIALS AND METHODSSynovial fluid, aspirated as part of a therapeutic procedure from knee joints of patients with rheumatoid arthritis, was put into sterile plastic containers and centrifuged at 10,OoOg for 20 minutes at 4°C. The cell-free supernatant synovial fluid was s...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.