Morales T I, Kuettner K E
Biochim Biophys Acta. 1982 Jul 12;705(1):92-101. doi: 10.1016/0167-4838(82)90340-5.
The enzymatic mechanism of proteoglycan breakdown is of major interest, since it has been proposed that osteoarthritis involves increased proteolytic breakdown of proteoglycans. This paper describes the properties of the proteoglycan-degrading enzymes released into the extracellular milieu by chondrocyte cultures that produce cartilage-specific type II collagen but no detectable type I collagen. Attention has been focused on enzymes active at neutral pH, since the pH of the extracellular matrix is around neutrality. Biogel P-60 chromatography of concentrated culture medium showed a major peak of enzyme activity on proteoglycan monomer entrapped in polyacrylamide beads as well as on native proteoglycan aggregates. The enzyme yields a specific limit digestion peptide from the aggregate of approximately 55,000 daltons (in the presence of SDS). This limit peptide is probably derived from the hyaluronic acid-binding region of proteoglycan. The proteolytic enzyme is latent but can be activated by aminophenylmercuric acetate or trypsin. The molecular weight of both the active and latent forms, determined by gel filtration, is approximately 33,000. The activity is not inhibited by phenylmethylsulfonyl fluoride or pepstatin but is completely inhibited by o-phenanthroline; the activity is restored by Zn or Co ions in the presence of calcium chloride. Removal of calcium by dialysis results in a reversible loss of activity. The release of such a metalloproteinase by chondrocytes into the extracellular milieu, its activity at physiological pH and its ability to degrade native proteoglycans are consistent with a role of the enzyme in proteoglycan metabolism.
蛋白聚糖分解的酶促机制备受关注，因为有人提出骨关节炎涉及蛋白聚糖蛋白水解分解增加。本文描述了由产生软骨特异性II型胶原蛋白但未检测到I型胶原蛋白的软骨细胞培养物释放到细胞外环境中的蛋白聚糖降解酶的特性。由于细胞外基质的pH值接近中性，因此注意力集中在中性pH下活性的酶上。浓缩培养基的Biogel P - 60色谱显示，在聚丙烯酰胺珠中截留的蛋白聚糖单体以及天然蛋白聚糖聚集体上有一个主要的酶活性峰。该酶从约55,000道尔顿的聚集体（在SDS存在下）产生特定的极限消化肽。这个极限肽可能来自蛋白聚糖的透明质酸结合区域。蛋白水解酶是潜伏性的，但可被氨基苯基汞乙酸盐或胰蛋白酶激活。通过凝胶过滤测定的活性和潜伏形式的分子量约为33,000。该活性不受苯甲基磺酰氟或胃蛋白酶抑制剂的抑制，但完全被邻菲罗啉抑制；在氯化钙存在下，锌或钴离子可恢复其活性。通过透析去除钙会导致活性可逆丧失。软骨细胞将这种金属蛋白酶释放到细胞外环境中，其在生理pH下的活性以及降解天然蛋白聚糖的能力与该酶在蛋白聚糖代谢中的作用一致。
