On the heterogeneity of the mitochondrial C27-steroid 26-hydroxylase system

Mitochondrial 26-hydroxylation of exogenous cholesterol, endogenous cholesterol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol was studied. 26-Hydroxylation of endogenous cholesterol was measured by mass fragmentography. NADPH and isocitrate stimulated 26-hydroxylation of endogenous as well as exogenous cholesterol. 26-Hydroxylation of endogenous cholesterol was linear with time for 15 min, whereas that of exogenous cholesterol was linear with time for at least 40 min. This finding indicates that the fractions of exogenous and endogenous cholesterol that were 26-hydroxylated did not equilibrate. Mg2+ stimulated isocitrate- and NADPH-dependent 26-hydroxylation of exogenous cholesterol but inhibited in the case of endogenous cholesterol. Ca2+ stimulated NADPH-dependent and inhibited isocitrate-dependent 26-hydroxylation of both exogenous and endogenous cholesterol. It is suggested that the differing effect of Mg2+ on the 26-hydroxylation of exogenous and endogenous cholesterol is related to transfer of the steroid to the enzyme. Isocitrate- and NADPH-dependent 26-hydroxylation of exogenous 5beta-cholestane-3alpha,7alpha,12alpha-triol differed from that of exogenous cholesterol in response to Mg2+ and Ca2+. 26-Hydrocylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol was stimulated by Mg2+ in low concentrations but inhibited by Mg2+ and Ca2+ in high concentrations. Mg2+ had the same influence on the 26-hydroxylation of three dioxygenated C27-steroids known to be intermediates in bile acid biosynthesis. The results are not only compatible with heterogeneity of the mitochondrial 26-hydroxylase system but also with differences in the transport of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to the enzyme. The finding of a differing effect of Mg2+ on 26-hydroxylation of exogenous and endogenous cholesterol seems to favor differences in transport rather than heterogeneity of the 26-hydroxylase as an explanation of the results.