Zeheb R, Rafferty U M, Rodriguez M A, Andreasen P, Gelehrter T D
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0618.
Thromb Haemost. 1987 Dec 18;58(4):1017-23.
Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45 degrees C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.
用合成糖皮质激素地塞米松孵育HTC大鼠肝癌细胞，可通过诱导一种特异性纤溶酶原激活物抑制剂（PAI-1）迅速抑制组织型纤溶酶原激活物活性。我们使用针对HT-1080人纤维肉瘤PAI-1制备的固定化多克隆抗体，从用地塞米松处理的HTC肝癌细胞条件培养液的无血清培养基中纯化了HTC PAI-1，并证明它与人类PAI-1在抗原性上相关。一步纯化即可实现大于100倍的纯化，产率大于75%。纯化后的PAI-1在SDS-聚丙烯酰胺凝胶上迁移时呈现出一条49 kDa的主要条带和一条46 kDa的次要条带。用内切糖苷酶F消化PAI-1会导致向迁移速度更快的物种转变，且这些物种保留抑制活性。纯化后的PAI-1在pH 2.5时稳定，在45℃下15分钟后失去50%的活性，在用SDS或盐酸胍处理后显示出明显的激活。纯化后的PAI-1能迅速抑制组织型和尿激酶型纤溶酶原激活物，并与之形成复合物。制备了多克隆兔抗大鼠PAI-1抗体，该抗体可免疫沉淀游离和复合形式的PAI-1。
