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

作　　者：杨挥[1] 张玲[1] 张旭家[1] 黄有国[1] 

机构地区：[1]生物大分子国家重点实验室

出　　处：《生物化学与生物物理进展》2001年第2期227-231,共5页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金!委重点项目 (39730 130 );中国科学院重大项目! (KJ95 1 A1 6 0 1)&&

摘　　要：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.

关 键 词：亚线粒体 Fo 重建 H+转运 纯化 质子转运 膜脂 

分 类 号：Q735[生物学—分子生物学]