Sorsa T, Suomalainen K, Turto H, Lindy S
Med Biol. 1985;63(2):66-72.
Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase.
从人多形核白细胞中提取了潜伏性和活性胶原酶。通过在琼脂糖6B上进行凝胶过滤来分离这两种酶形式。在用胰蛋白酶短暂处理或使各组分暴露于巯基试剂氯化苯汞后，从色谱级分中检测到酶的潜伏形式。潜伏性酶比活性酶先从柱上洗脱下来，表明其表观分子量更高。通过凝胶过滤估计，部分纯化的潜伏性酶的表观分子大小为70 - 75 kDa。活性酶的该值为50 - 55 kDa。未经激活，潜伏性酶不会降解可溶性胶原底物。这通过定量粘度测定法以及十二烷基硫酸钠聚丙烯酰胺凝胶电泳得到了证实，此时看不到典型的胶原裂解产物。除非在提取和纯化过程中存在丝氨酸蛋白酶抑制剂，否则无法获得潜伏性酶。研究了激活剂胰蛋白酶、氯化苯汞、苯甲基磺酰胰蛋白酶和N - 乙基马来酰亚胺对潜伏性人多形核白细胞胶原酶的作用。与无活性蛋白酶激活潜伏性人多形核白细胞胶原酶的观点相反，无活性的苯甲基磺酰胰蛋白酶不能激活潜伏性胶原酶。
