Sueishi K, Nanno S, Okamura T, Inoue S, Tanaka K
Biochim Biophys Acta. 1982 Aug 6;717(2):327-36. doi: 10.1016/0304-4165(82)90187-8.
The tissue activator was extracted with 2 M ammonium thiocyanate and purified by L-arginine methyl ester, concanavalin A and ion-exchange chromatographies, and Sephacryl S-200 gel filtration in buffers containing Triton X-100 and/or ammonium thiocyanate. The final preparations had specific activities of 25 000-40 000 IU/mg protein and were shown to be a single band with an apparent molecular weight of 54 00 by SDS-polyacrylamide gel electrophoresis with or without reducing agent. When subjected to isoelectric focusing, its major component had an isoelectric point of approx. 8.2 with minor components. (7.8-8.6). The purified tissue activator was a serine protease, dissimilar to urokinase in some respects including antigenicity, strong affinity to insoluble fibrin monomer and hydrolytic activities for synthetic substrates. The crude extract contained another plasminogen activator with antigen identity to urokinase, which constituted approx. 15% of the total activity in crude extract. These findings indicated that human kidney would produce at least two plasminogen activators, namely, the tissue activator as a major plasminogen activator and urokinase.
用2M硫氰酸铵提取组织激活剂，并通过L-精氨酸甲酯、伴刀豆球蛋白A和离子交换色谱法，以及在含有 Triton X-100和/或硫氰酸铵的缓冲液中进行Sephacryl S-200凝胶过滤进行纯化。最终制剂的比活性为25000-40000 IU/mg蛋白质，在有或没有还原剂的情况下，通过SDS-聚丙烯酰胺凝胶电泳显示为一条单一的条带，表观分子量为5400。进行等电聚焦时，其主要成分的等电点约为8.2，还有一些次要成分（7.8-8.6）。纯化的组织激活剂是一种丝氨酸蛋白酶，在某些方面与尿激酶不同，包括抗原性、对不溶性纤维蛋白单体的强亲和力以及对合成底物的水解活性。粗提物中含有另一种与尿激酶具有抗原同一性的纤溶酶原激活剂，约占粗提物总活性的15%。这些发现表明，人肾至少会产生两种纤溶酶原激活剂，即作为主要纤溶酶原激活剂的组织激活剂和尿激酶。
