Cytochrome P-450scc induces vesicle aggregation through a secondary interaction at the adrenodoxin binding sites (in competition with protein exchange)

Abstract

Addition of bovine adrenal cytochrome P-450scc to small unilamellar dioleoylphosphatidylcholine vesicles (DOPC-SUV) produces a complex sequence of interactions, indicating exceptional cytochrome mobility. First, cholesterol transfer from cytochrome to vesicles indicated rapid dissociation of P-450scc oligomers and integration of monomers into the membrane (delta A 390-420 nm; t1/2 = 2 s). After 10-15 s, P-450scc-induced aggregation of the vesicles starts, as indicated by increased turbidity (delta A 448 or 520 nm; complete in 6-8 min). Fluorescence quenching experiments indicate that this aggregation does not lead to measurable vesicle fusion during this period. Aggregation is prevented by mild heat denaturation of P-450scc, by addition of anti-P-450scc IgG, and also by 1:1 complex formation with the electron donor adrenodoxin (ADX). P-450scc, therefore, links two vesicles through two separate domains involved in, respectively, membrane integration (lipophilic) and ADX binding (charged). Although completely bound by DOPC-SUV, as evidenced by Sephadex elution, P-450scc has access within 1 min to cholesterol in secondary SUV. This is indicated by spectral changes (cholesterol complex formation) and by metabolism of secondary vesicle cholesterol. Since cholesterol equilibrates slowly between vesicles (t1/2 = 1-2 h), these changes arise from P-450scc transfer. This transfer was maximally slowed after a 5-min preincubation with primary vesicles, reflecting more extensive integration into the membrane than is necessary for the rapid initial cholesterol transfer to P-450scc. P-450scc transfer probably results from simultaneous interaction of P-450scc with two vesicles that may also initiate aggregation. Weaker integration into primary dimyristoylphosphatidylcholine vesicles facilitates exchange but prevents aggregation. Integration and aggregation are both enhanced by incorporation of 10% phosphatidylinositol into SUV, while exchange is slowed. This mobility of P-450scc is most probably a consequence of the absence of amino-terminal anchoring. P-450scc-induced association of inner mitochondrial membrane segments may contribute to the exceptionally vesiculated structure of adrenal and ovarian mitochondria that parallels increased P-450scc content.

Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

“…6). These results are consistent with the activity of P-450scc being determined by the concentration of cholesterol in the membrane, as has been established previously (25,29,31,34). For a given amount of cholesterol transferred, P-450scc activity decreases as the ratio of cholesterol to phospholipid decreases because of an increase in the concentration of acceptor phospholipid.…”

“…The results of our study and those of others (34) show that there is little vesicle fusion over the typical time period used in the current study (34). P-450scc spontaneously incorporates into these vesicles with its adrenodoxin-binding site facing the exterior (28,31,37).…”

“…From two replicate experiments the mean V max was 22.4 pmol of pregnenolone/min/pmol of P-450scc, and the mean K m was 0.070 mol of cholesterol/mol of phospholipid. Measurement of Phospholipid, Triglyceride, Transfer from Acceptor to Donor Vesicles-To measure transfer between vesicles, neutral donor vesicles comprising dioleoyl phosphatidylcholine and 0.2 mol of cholesterol/mol of phospholipid were separated from acidic acceptor vesicles comprising dioleoyl phosphatidylcholine, 15 mol % cardiolipin, and 0.03 M P-450scc by chromatography on DEAE-Sepharose CL-6B in a method comparable with that used by other workers (23,(32)(33)(34). Acceptor vesicles were labeled with 2 nCi of di[1-…”

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

“…Although earlier reports denied P450SCC exchange in soy PC vesicles (15), later such an exchange was found for DOPC vesicles and explained by the specific capacity of P450SCC to bridge between two vesicle membranes thereby using two distinct domains on the protein (3). One domain must be at least partially overlap with the adrenodoxin binding region, probably involving positively charged regions on P450SCC (3,24). Note that we never observed a lag time for metabolism in secondary DOPC vesicles as reported for small unilamellar DOPC vesicles (3).…”

“…[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).…”