The specificity of the rat-liver cholesterol 7alpha-hydroxylase.

Abstract

Experiments have been performed which investigate the relationship of the cholesterol 7α‐hydroxylase enzyme to the cytochrome‐P ‐450‐dependent mixed‐function oxidase system of rat‐liver microsomes. There appears to be no simple relationship between the activity of the cholesterol 7α‐hydroxylase enzyme and the cytochrome P ‐450 content of rat liver microsomes. Rat liver microsomal cholesterol 7α‐hydroxylase was sensitive to cycloheximide treatment. Its activity decayed in vivo following this treatment more rapidly than the aminopyrine demethylase enzyme or the mixed‐function oxidase components cytochrome P ‐450 and NADPH‐cytochrome c reductase. Freezing and thawing of rat liver microsomes resulted in a marked decrease in the activity of the cholesterol 7α‐hydroxylase.The specificity of the cholesterol 7α‐hydroxylase enzyme has been investigated by comparing the liver microsomal oxidation of cholesterol with that of other steroids having a similar structure to cholesterol.The substrates whose oxidation was studied were androst‐5‐en‐3β‐ol‐17‐one, pregn‐5‐en‐3β ol‐20‐one, androst‐5‐en‐3β‐ol and pregn‐5‐en‐3β‐ol. All these steroids were metabolised to one or more products when incubated with rat liver microsomes, NADPH and oxygen. Treatment of rats with cholestyramine which increased the activity of cholesterol 7α hydroxylase, had no effect on the rate of oxidation of any of the four steroids studied.The conversion of [4‐14C]cholesterol to 7α‐hydroxy[4‐14]cholesterol was significantly increased by preincubating the added tracer cholesterol with liver microsomes prior to starting the hydroxylation reaction with NADPH.It is suggested that the observed differences between the mechanism of control of the cholesterol 7α‐hydroxylase enzyme and other mixed‐function oxidase enzymes in rat liver microsomes might be due to presence of different rate‐limiting steps in the overall enzyme complex.