Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: XXVI. SPECIFICITY OF PHOSPHOLIPIDS REQUIRED FOR ENERGY TRANSFER REACTIONS

Abstract

Abstract 1. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven proton translocation were reconstituted with chemically defined phospholipids and mitochondrial membrane proteins which were virtually free of electron transport carriers. 2. Phosphatidylcholine and phosphatidylethanolamine were both required for the reconstitution of vesicles with high exchange activity. The optimal ratio of the two phospholipids was variable depending on the source and composition of the phospholipids. With highly purified preparations a 3- to 4-fold excess of phosphatidylethanolamine over phosphatidylcholine yielded optimal rates. An equal molar ratio gave low exchange activity, which was markedly accelerated by low amounts of cardiolipin or another acidic phospholipid. Coenzyme Q10 was not required. 3. Synthetic preparations substituted for natural phospholipids. The presence of unsaturated fatty acyl groups in the phospholipid appeared to be essential for the reconstitution of active vesicles. Phospholipids with fully saturated acyl groups were actually inhibitory. On the other hand, phospholipids with unnatural side chains were active provided they contained unsaturated groups. 4. The morphology of the reconstituted vesicles as seen in electron micrographs varied with the different phospholipids used in reconstitution. 5. Optimal conditions of reconstitution of the components which were solubilized with cholate were studied with [carboxy-14C]cholate. Rapid removal of cholate by passage of the components through a Sephadex column resulted in inactive particles. Removal of cholate by dialysis over several hours was optimal. The reconstituted vesicles had a higher phospholipid content and lower proton permeability than sub-mitochondrial particles.