Competition for electron transfer between cytochromes P450scc and P45011 beta in rat adrenal mitochondria.
Rat adrenal mitochondria contain approximately equal levels of P450scc and P45011 beta, each reduced by NADPH through adrenodoxin reductase (ADX-reductase) and adrenodoxin (ADX). Constitutive cholesterol side-chain cleavage (SCC) can be increased over 20-fold through a combination of hormonal activation and inhibition of cholesterol metabolism in vivo prior to isolation of the mitochondria. This stimulation, which results from accumulated reactive cholesterol, does not significantly affect either the dependence of activities on the concentration of isocitrate (IC) and succinate (SU) or the ratio of maximum activities [3:1] supported by these reductants. Thus, the rate of cholesterol SCC is determined independently by electron transfer and the amount of reactive cholesterol. Hydroxylation of deoxycorticosterone (11 beta and 18 positions) required much higher levels of each reductant, indicating less effective reductant transfer to P45011 beta. Reactions at P450scc and P45011 beta, mediated by IC, are enhanced by low concentrations of various dicarboxylates anions (fumarate, SU). The actions of SU dehydrogenase inhibitors and the activity of fumarate, a poor direct reductant, suggest that higher production of NADPH results from malate-enhanced uptake of isocitrate. Only synergistic combinations of reductants are sufficient to sustain maximum rates of 11-deoxycorticosterone (DOC) metabolism, whereas IC is fully effective for P450scc. Increased reaction at P450scc (cholesterol loading or addition of 20 alpha-hydroxycholesterol) decreased simultaneous DOC metabolism at P45011 beta in inverse proportion to the estimated intramitochondrial generation of NADPH (1 mM or 10 mM SU > 1 mM IC > 10 mM IC). These decreases were reversed by inhibition of P450scc. Crossover inhibition caused by maximum DOC metabolism was less pronounced. EGTA/albumin treatment, which enhanced activities at both P450scc and P45011 beta, presumably via increased NADPH, diminished this cross-competition. The differential dependence on reductants and the characteristics of crossover competition are consistent with a roughly three-fold more favorable partitioning of electron transfer to P450scc, possibly caused by preferential interaction of reduced adrenodoxin with P450scc.
Yamazaki
T
T
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
McNamara
B C
BC
Jefcoate
C R
CR
D000302:Adrenal Cortex / Q000201:enzymology* / Q000648:ultrastructure; D000325:Adrenodoxin / Q000378:metabolism; D000616:Aminoglutethimide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D003366:Cosyntropin / Q000494:pharmacology; D003900:Desoxycorticosterone / Q000378:metabolism; D004579:Electron Transport; D005260:Female; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D007523:Isocitrates / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008797:Metyrapone / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011284:Pregnenolone / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism*; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid
D007523:Isocitrates; D007527:Isoenzymes; D013386:Succinates; D000616:Aminoglutethimide; D000325:Adrenodoxin; C026416:aminoglutethimide phosphate; D003366:Cosyntropin; D003900:Desoxycorticosterone; D009249:NADP; D011284:Pregnenolone; D002784:Cholesterol; C034219:isocitric acid; D019802:Succinic Acid; D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D008797:Metyrapone