猪心线粒体F_o的纯化、重建及其质子转运功能

Purification, Reconstitution and H^+ Translocation Activity of Porcine Heart Mitochondrial F_o

比较了猪心线粒体FoF1 ATPase膜部分Fo 的四种纯化方法 .结果表明 ,用NaBr从亚线粒体除去FoF1 ATPase的水溶性部分F1 ATPase后 ,再以CHAPS增溶 ,并经蔗糖梯度离心 ,可获得高纯度的Fo.SDS 聚丙烯酰胺凝胶电泳鉴定表明 ,纯化的Fo 含有b、OSCP (寡霉素敏感授予蛋白 )、d、a、e、F6、IF1、A6L和c等 9种亚基 .用去污剂稀释法将纯化的Fo 在脂质体上重建后 ,重建Fo 表现较高的被动转运质子活性 .这为在体外深入研究Fo 的活性、构象与膜脂的关系 ,以及Fo 与F1 ATPase的组装等提供了很好的实验模型 .

Four methods were compared to purify F-o from porcine heart mitochondria. The best results were obtained by the following method: after removing F-1-ATPase with NaBr incubation from submitochondrial FoF1-ATPase, F-o was solubilized with CHAPS and purified by sucrose density gradient centrifugation. SDS-PAGE with silver staining showed about 85% purity of the isolated F-o and 9 different subunits including b, OSCP, d, a, e, F-6, IF1, A6L and c. The purified F-o was then incorporated into asolectin liposomes, the reconstituted F-o showed higher H+ translocation activity and after F-o was reconstituted with F-1-ATPase, the resulted FoF1- ATPase complex exhibited high ATP hydrolysis activity and high sensitivity to oligomycin. The results provide evidence for successful purification, reconstitution of F-o with high H+ translocation activity and its relationship with phospholipids.