Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles

Abstract

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.

Branched Phosphatidylcholines Stimulate Activity of Cytochrome P450SCC (CYP11A1) in Phospholipid Vesicles by Enhancing Cholesterol Binding, Membrane Incorporation, and Protein Exchange

“…in the order PC(14,10) Ͼ PC(12,8) Ͼ PC(10,6). The observed rates (V max Ͼ 30 min Ϫ1 ) are only comparable with the highest rates reported so far for micellar systems using Tween 20 as detergent (17,19) and for DOPC vesicle systems prepared by octylglucoside dialysis (2). In the latter case the high activity results from residual octylglucoside.…”

“…Both compounds seem to impact the membrane with the same structural property leading to activation of P450SCC via enhancement of CHL binding to P450SCC. Biphasicity in activity is not new; it has been already reported earlier for octylglucoside-reconstituted DOPC vesicles and interpreted as resulting from residual octylglucoside (2). An additional second phase of complex formation also has been reported for DMPC/CL vesicles (7).…”

“…[2][3][4]. Under the lipids used in reconstitution CL 1 played a special role mainly because (i) it is a typical mitochondrial lipid and (ii) it represents the most potent activator of P450SCC activity (5)(6)(7).…”

α-Branched 1,2-Diacyl Phosphatidylcholines as Effectors of Activity of Cytochrome P450SCC (CYP11A1)

“…To study the chain dependence in greater detail, it would be interesting to use branched PCs with even longer acyl chain substituents in the 2-position; such experiments are now under way. (10,6) or PC (12,8) P450SCC-lipid interaction, vesicles made on the basis of DOPC were often used; applying these a rather high rate of enzymatic activity as compared to DMPC could be observed (1,19,22). Thus, it may be of interest to see to what extent ␣-branched PCs are capable of exerting their influence on the various properties of P450SCC reconstituted into vesicles from DOPC.…”

“…Furthermore, it has been reported previously that reconstitution by both octylglucoside and cholate dialysis results in the formation of two main populations of vesicles, larger and smaller ones with the smaller vesicles containing significantly more P450SCC (31,32). Recently it was reported that the size of the vesicles, which in turn determines the vesicle curvature, has a large influence on the activity (22). Taken together, these findings all suggest that the curvature of the membrane plays a role for the incorporation of P450SCC and thus support our view that polymorphic lipids like CL and the branched PCs could influence P450SCC by maintenance of the membrane curvature at a value optimal for P450SCC activity.…”