Side-chain cleavage of cholesterol sulfate by ovarian mitochondria

Abstract

Mitochondria isolated from porcine corpora lutea and from the luteinized ovaries of gonadrotropin-treated immature rats were found to efficiently cleave the side-chain of cholesterol sulfate to produce 3β-hydroxy-5-pregnen-20-one sulfate (pregnenolone sulfate). When mitochondria were preincubated with cholesterol sulfate, the time-course for the side-chain cleavage of cholesterol sulfate was biphasic. With 200 μM cholesterol sulphate, the initial rate of the reaction was the same as that observed for 25-hydroxycholesterol. This rate was not increased when both cholesterol sulfate and 25-hydroxycholesterol were incubated together. The rate of side-chain cleavage by isolated mitochondria supplied with 75 μM cholesterol sulfate as substrate was inhibited by 97% by aminoglutethimide, a specific inhibitor of cytochrome P-450 scc. The slow phase of side-chain cleavage of cholesterol sulfate appeared to be limited by the rate of substrate movement to the mitochondrial site of the reaction. Cholesterol sulfate translocation rates were however up to 8 times greater than those observed for cholesterol when equivalent concentrations of the two substrates were added to the mitochondria. We conclude that cholesterol sulfate is a better substrate than cholesterol for side-chain cleavage by isolated mitochondria and that both reactions are catalysed by the same cytochrome P-450 scc enzyme.