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The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.

ABSTRACT: 1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.

SUBMITTER: Sellers A

PROVIDER: S-EPMC1162032 | biostudies-other | 1980 Sep

REPOSITORIES: biostudies-other

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Project description:The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.

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The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.

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