title: Conversion of cholesterol to pregnenolone mobilizes cytochrome P-450 in the inner membrane of adrenocortical mitochondria: protein rotation study.
abstract: Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Institute of Physics, College of Arts and Sciences, University of Tokyo at Komaba.
authors.forename: Y
authors.initials: Y
authors.lastname: Ohta
authors.forename: F
authors.initials: F
authors.lastname: Mitani
authors.forename: Y
authors.initials: Y
authors.lastname: Ishimura
authors.forename: K
authors.initials: K
authors.lastname: Yanagibashi
authors.forename: M
authors.initials: M
authors.lastname: Kawamura
authors.forename: S
authors.initials: S
authors.lastname: Kawato
mesh_terms: D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D004058:Diffusion; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005454:Fluorescence Polarization; D005615:Freezing; D007425:Intracellular Membranes / Q000201:enzymology*; D008560:Membrane Fluidity; D008928:Mitochondria / Q000201:enzymology*; D008956:Models, Chemical; D011284:Pregnenolone / Q000096:biosynthesis*
chemical_list: D000325:Adrenodoxin; D011284:Pregnenolone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase