Cardiolipin is essential for higher proton translocation activity of reconstituted F0

The F₀ membrane domain of F₀F₁-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F₀ was about 85% and the sample contained no subunits of F₁-ATPase. The purified F₀ 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 F₀ was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F₀ with the order of CLPA>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F₀ vesicles containing CL, which suggested that CL’s stimulation of the activity of reconstituted F₀ might correlate with its non-bilayer propensity. After F₀ was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F₀ 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 F₀ at cysteine indicated that CL could induce F₀ to a suitable conformation resulting in higher proton translocation activity.