Cytochrome P-450scc induces vesicle aggregation through a secondary interaction at the adrenodoxin binding sites (in competition with protein exchange.
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.
Dhariwal
M S
MS
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Kowluru
R A
RA
Jefcoate
C R
CR
D000311:Adrenal Glands / Q000201:enzymology; D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D002850:Chromatography, Gel; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D013053:Spectrophotometry
D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D000325:Adrenodoxin; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; C017251:1,2-oleoylphosphatidylcholine