Melrose J, Ghosh P, Taylor T K
Biochim Biophys Acta. 1987 Mar 19;923(3):483-95. doi: 10.1016/0304-4165(87)90058-4.
Disc tissue consisting of pooled annuli fibrosus and nuclei pulposus from the cadaver of an adolescent aged 19 years was extracted with 4.0 M Gu-HCl. Proteins of low buoyant density (p less than or equal to 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density (p greater than or equal to 1.50 g/ml) by density gradient ultracentrifugation. Sephadex G-75F gel chromatography followed by trypsin affinity chromatography was then used to resolve disc proteolytic and trypsin inhibitory activities. The results obtained were strongly suggestive of the presence of a high molecular weight zymogen which upon activation generated a population of smaller molecular weight proteinases. The disc proteinases obtained by this process showed similar properties in terms of: their pH optima (7.4-7.6); their inhibition patterns by class-specific proteinase inhibitors; their variation of activity as a function of NaCl and lysine concentrations; and the hydrodynamic size of their proteoglycan degradation products. The activated disc neutral proteinase demonstrated many characteristics in common with plasmin; however, unlike the latter, the disc proteinases also showed some calcium dependence.
从一名19岁青少年尸体中获取的由纤维环和髓核混合而成的椎间盘组织，用4.0 M盐酸胍进行提取。通过密度梯度超速离心，将含有椎间盘酶和抑制剂的低浮力密度（p≤1.38 g/ml）蛋白质与高浮力密度（p≥1.50 g/ml）的蛋白聚糖分离。随后采用葡聚糖G - 75F凝胶色谱法，接着进行胰蛋白酶亲和色谱法，以解析椎间盘的蛋白水解活性和胰蛋白酶抑制活性。所获得的结果强烈表明存在一种高分子量的酶原，该酶原激活后会产生一群分子量较小的蛋白酶。通过此过程获得的椎间盘蛋白酶在以下方面表现出相似的特性：它们的最适pH值（7.4 - 7.6）；它们被类别特异性蛋白酶抑制剂的抑制模式；它们的活性随氯化钠和赖氨酸浓度的变化；以及它们蛋白聚糖降解产物的流体动力学大小。激活后的椎间盘中性蛋白酶表现出许多与纤溶酶相同的特征；然而，与纤溶酶不同的是，椎间盘蛋白酶还表现出一定的钙依赖性。
