The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.
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.
D000818:Animals; D001391:Azo Compounds; D002356:Cartilage / Q000032:analysis; D003094:Collagen; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007743:Labor, Obstetric; D008666:Metalloendopeptidases; D008667:Metalloproteins; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014599:Uterus / Q000201:enzymology*
D001391:Azo Compounds; D008667:Metalloproteins; D011480:Protease Inhibitors; D011509:Proteoglycans; C028470:Azocoll; D003094:Collagen; D010450:Endopeptidases; D008666:Metalloendopeptidases