Cardiolipin is essential for higher proton translocation activity of reconstituted F_o

The Fo membrane domain of FoF1-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL>PA>>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphospha     

心磷脂对重建Fo的质子转运高活性是必需的

从猪心线粒体纯化了F_oF₁-ATPase的膜部分质子通道F_o,SDS-PAGE表明纯度约85%,且不含F₁-ATPase的各亚基.将纯化的F_o在含不同纯磷脂组分的脂质体上重建,分别测定心磷脂(CL)、磷脂酸(PA)、磷脂酰肌醇(PI)和磷脂酰丝氨酸(PS)等酸性磷脂对重建F_o质子转运活性的影响,结果表明,它们促进重建F_o质子转运活性的强弱顺序是CL＞PA＞＞ PI,而PS却完全抑制重建F_o的质子转运.阿霉素(ADM)显著抑制含CL的重建F_o的质子转运活性,提示CL明显促进F_o的质子转运可能与其具有强烈的形成非双层脂结构的倾向性相关.测定磷脂酰乙醇胺(PE)及二顺油酰磷脂酰乙醇胺(DOPE)、二反油酰磷脂酰乙醇胺(DEPE)对重建F_o质子转运活性的影响表明,有形成非双层脂结构倾向性的PE,DOPE明显促进重建F_o的质子转运.荧光猝灭剂HB对重建F_o的内源荧光猝灭及专一性标记蛋白质巯基的荧光探剂acrylodan的荧光光谱分析进一步表明,适当浓度的CL可能使重建F_o具有适合的构象,并表现高的质子转运活性.     

猪心线粒体F_o的纯化、重建及其质子转运功能

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C HAPS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.     

猪心线粒体Fo的纯化、重建及其质子转运功能

比较了猪心线粒体F_oF₁-ATPase膜部分F_o的四种纯化方法.结果表明,用NaBr从亚线粒体除去F_oF₁-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F_o.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的F_o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F_o在脂质体上重建后,重建F_o表现较高的被动转运质子活性.这为在体外深入研究F_o的活性、构象与膜脂的关系,以及F_o与F₁-ATPase的组装等提供了很好的实验模型.