Characterization of cytochrome P-450scc-containing liposomes

Purified cytochrome P450scc from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of P450scc into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When P450scc was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the P450scc-containing liposomes showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200-600 A and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, P450scc was less stable than P450scc in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal P450scc. Liposomal P450scc required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal P450scc was subjected to p-chloromercuriphenyl sulfonic acid treatment. This reagent destroyed the liposomal P450scc. These results suggest that the heme is located in the proximity of the p-chloromercuriphenyl sulfonic acid reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.