Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.
Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.
D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000032:analysis; D007700:Kinetics; D008081:Liposomes; D008928:Mitochondria / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011485:Protein Binding; D013329:Structure-Activity Relationship
D005227:Fatty Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme