Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles.
Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome.
Dhariwal
M S
MS
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Jefcoate
C R
CR
D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D008081:Liposomes; D008854:Microscopy, Electron; D010743:Phospholipids; D013010:Sonication; D013229:Stearic Acids / Q000494:pharmacology*
D005960:Glucosides; D006027:Glycosides; D008081:Liposomes; D010743:Phospholipids; D013229:Stearic Acids; C018619:octyl-beta-D-glucoside; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol