The Journal of Steroid Biochemistry and Molecular Biology 1992-09-01

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates.

R C Tuckey

Index: J. Steroid Biochem. Mol. Biol. 42(8) , 883-90, (1992)

Full Text: HTML

Abstract

The side-chain cleavage of cholesterol by cytochrome P-450scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20 alpha-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20 alpha,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20 alpha,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450scc-20 alpha,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450scc-20 alpha,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450scc occur at approximately the same rate.