Cardiolipin is essential for higher proton translocation activity of reconstituted F(0)

The F(0) membrane domain of F(0)F(1)-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F(0) was about 85% and the sample contained no subunits of F(1)-ATPase. The purified F(0) 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 F(0) was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F(0) with the order of CLPA>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F(0) vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted F(0) might correlate with its non-bilayer propensity. After F(0) was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F(0) vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling F(0) at cysteine indicated that CL could induce F(0) to a suitable conformation resulting in higher proton translocation activity.