Woessner J F, Selzer M G
J Biol Chem. 1984 Mar 25;259(6):3633-8.
Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.
人类关节软骨中的金属蛋白酶活性水平极低；1克软骨中的活性大约相当于1微克胰蛋白酶的活性。基于放射性蛋白聚糖消化的灵敏检测方法的开发，使得研究1 - 2克软骨标本中的蛋白酶活性成为可能。软骨在低温下用Tris缓冲液提取，并在60℃下用含有10 mM氯化钙的Tris缓冲液提取。提取物通过琼脂糖6B；检测到两种主要和两种次要的金属蛋白酶活性。发现一种最适pH为7.4的中性金属蛋白酶活性以Mr = 56,000的潜在形式存在。它可以用乙酸氨基苯汞或胰蛋白酶激活，结果Mr降至40,000。一种最适pH为5.3的酸性金属蛋白酶也以Mr = 50,000的潜在形式存在。激活后其变为Mr = 35,000。通过透析去除钙离子可使中性酶的活性降低80 - 85%，酸性酶的活性降低100%。10 mM Ca2+可恢复两种活性。在过量钙离子存在下，两种酶均被1 mM邻菲罗啉完全抑制。1 mM Zn2+可克服这种抑制，Co2+在较小程度上也可克服。这些蛋白酶可能在软骨基质的代谢及其在骨关节炎中的破坏中起重要作用。
