A study of proteases and protease-inhibitor complexes in biological fluids.
A study of proteases and protease-inhibitor complexes in biological fluids.
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DOI：
10.1084/jem.148.1.223
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发表时间：
1978-07-01
期刊：
影响因子：
--
通讯作者：
Reich E
中科院分区：
文献类型：
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作者：
Granelli-Piperno A;Reich E
我们（a）筛选了多种细胞系和体液中的纤溶酶原激活剂，（b）在将复合物暴露于部分降解和/或变性程序以揭示蛋白水解活性后，研究了与α2-巨球蛋白结合的蛋白酶的活性。各自的结果表明：(a)尿液和细胞培养基中的纤溶酶原激活剂的分子量通常低于血浆中的分子量； (b) 与 α2-巨球蛋白结合的蛋白酶在暴露于十二烷基硫酸钠后恢复了攻击大分子底物的能力，同时保留了蛋白酶抑制剂复合物的电泳迁移率。这表明蛋白酶和抑制剂可能通过共价键连接。相比之下，蛋白酶和较低分子量抑制剂（例如大豆或 Kunitz 抑制剂）之间形成的其他复合物可被十二烷基硫酸钠 (SDS) 完全解离。所描述的实验基于检测 SDS 聚丙烯酰胺凝胶中蛋白水解酶活性的新程序。该方法依靠非离子去污剂溶液提取 SDS，然后将电泳凝胶涂在由纤维蛋白-琼脂混合物组成的指示凝胶上。该方法灵敏，可检测不到 1 μl 新鲜血浆中的蛋白酶，并且可有效解决分子量的微小差异。该程序可以定量，并且经过针对每个特定系统的微小修改，它已应用于广泛的丝氨酸酶和酶原，包括一些在纤维蛋白溶解、凝血和激肽生成途径中起作用的丝氨酸酶和酶原。其他潜在的应用似乎也是可能的。
We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely.