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Abstract
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.
Highlights
ROLE OF T H E MEMBRANE IN CONTROL OF ACTIVITY AND SPIN STATE OF T H E CYTOCHROME*
Cytochrome P-450, from bovine adrenal cortexmi- transfer in this system and find that adrenodoxin acts as a tochondria was purified and reconstituted into phos- mobile one-electron shuttle between adrenodoxin reductase phatidylcholine vesicles which varied in both choles- and cytochromeP-450, [4,5,6]; the adrenodoxin must interact terol content and in the fatty acyl composition of the with an aqeuous-exposed site on the cytochromeonce during phospholipid
Whencholesterol is incorporated into the phospholipid membrane, binding of cholesterol to the cytochrome occurs rapidly and is accompanied by a conversion of the optical spectrum from a low spin or L-type spectrum tao high spinor H-type spectrum[9].The fraction of hemoprotein of differing fatty acyl composition showed an excellent in the high spin form ias function of the cholestero1:phosphoinverse correlation with the rateosf pregnenolone for- lipid ratioratherthanthetotal cholesterol concentration mation in the same lipids; phospholipid exerts its adopted the convention of expressing cholesterol concentrarate effects by modulating the binding of cholesterol to tion as a cholestero1:phospholipid molarratio
Summary
Glucose 6-phosphate, pure egg phosphatidylcholine, and glucose-6-phosphate dehydrogenase were obtained from Sigma; NADPH was purchased from P-L Biochemicals; and [7-“Hlpregnenolone (5-pregnen-3/&ol-20-one),. “C]tripalmitin were products of New England Nuclear. Hexyl agarose was from Miles-Yeda and antipregnenolone antibody was from Radioassay Systems Laboratories. Cholesterol was purchased from Applied Science Laboratories, and contained less than 1% contamination by gas chromatography and thin layer chromatography. Dimyristoylphosphatidylcholine was from Calbiochem, and all other phospholipids including hydrogenated egg phosphatidylcholine were purchased from Avanti Biochemicals. Cholic acid was purified nrior to use bv charcoal treatment and recrystallization from hot 95% ethanol
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