Enzymatic Characteristics of CO‐Sensitive 26‐Hydroxylase System for 5β‐Cholestane‐3α, 7α, 12α‐triol in Rat‐Liver Mitochondria and Its Intramitochondrial Localization

Abstract

The enxymatic characteristics of mitochondrial 26-hydroxylase system for 5β-cholestane-3α,7α,12α-triol were studied with rat liver mitochondria and the inner membrane-matrix fractions with special reference to its intramitochondrial localization. 1 Conditions for the assay of enzymatic activity of the hydroxylase system were established with rat liver mitochondrial preparations and inner membrane-matrix fractions derived by digitonin treatment. 2 It was established that rat liver mitochondria, as well as microsomes, possessed the hydroxylase system for 5β-cholestane-3α,7α,12α-triol as an intrinsic constituent. Product analyses revealed that only the mitochondrial hydroxylase system was specifically active for C-26 position. In contrast, the microsomal system was found to be apparently unspecific on C-26 position but rather more active for other adjacent positions. 3 Among the tested citric acid cycle intermediates and nicotinamide coenzymes, isocitrate and NADPH exerted the most remarkable enhancement of the 26-hydroxylase activity. NADP and NADH were totally inert under the present assay conditions. 4 Partial rupture of the mitochondrial structure by hypotonic or digitonin treatment was essential for the reactivity of 26-hydroxylase system under the present experimental conditions. Accessibility to the intramitochondrial reacting site of the exogenous reactants such as NADPH, isocitrate and 5β-cholestane-3α,7α,12α-triol in particular was apparently improved by the partial rupture. 5 The NADPH-dependent or isocitrate-dependent 26-hydroxylase system was totally insensitive to inhibitors of the respiratory electron transfer such as potassium cyanide, antimycin A, rotenone and amytal. However, it was specifically inhibited by phenyl isocyanide. 6 Remarkable sensitivity to carbon monoxide of the 26-hydroxylase system was demonstrated. Apparent Michaelis constant for oxygen and partition coefficient (between carbon monoxide and oxygen) of the 26-hydroxylase system were estimated to be approximately 10–20 μM and 0.1, respectively. Thus, the possible functioning of a “cytochrome P-450”-like entity in intramitochondrial 26-hydroxlase system was proposed. 7 The localization of 26-hydroxylase system was assigned to be in the inner membranematrix region, on the basis of the distribution of intramitochondrial marker enzymes during the course of serial solubilization by digitonin.