Mitochondrial omega hydroxylation of cholesterol side chain

The mitochondrial fraction of rat liver homogenate catalyzed the conversion of cholesterol into 5 cholestene 3β,25 diol and 5 cholestene 3β,26 diol in the presence of an NADPH generating system and oxygen. 5 Cholestene 3β,26 diol was the predominant product. 26 Hydroxylation of cholesterol was much more efficient with the NADPH generating system than with NADPH alone, and it was shown that the isocitrate in the generating system was responsible for most of the stimulation. Addition of Mg²⁺ increased both the NADPH and the isocitrate dependent 26 hydroxylation, and at high concentrations of Mg²⁺ the 26 hydroxylation in the presence of NADPH was about the same as in the presence of isocitrate. Addition of Ca²⁺ increased NADPH dependent but not isocitrate dependent 26 hydroxylation. NADH and NADH generating systems could not replace NADPH or the NADPH generating system. However, NADH together with Mg²⁺ and ATP stimulated the reaction efficiently, probably due to energy dependent intramitochondrial transhydrogenation. Citrate and isocitrate were found to stimulate mitochondrial 26 hydroxylation of cholesterol more efficiently than other citric acid cycle intermediates regardless of whether Mg²⁺ was present or absent. Mitochondrial 26 hydroxylation was inhibited by p hydroxymercuribenzoate. Rotenone, potassium cyanide, and dinitrophenol had little or no effect on isocitrate dependent 26 hydroxylation. Hg²⁺, Zn²⁺, Cu²⁺, and Fe³⁺ in 0.1 mM concentration inhibited the reaction strongly. Experiments with sonically disrupted mitochondria supported the contention that the mitochondrial 26 hydroxylase was mainly bound to the inner membranes. Experiments with ¹⁸O₂ and ²H₂O showed that in 25 as well as 26 hydroxylation of cholesterol the oxygen incorporated is derived from molecular oxygen.