A simplified assay of 4-methyl sterol oxidase of liver microsomes

Abstract

Microsomal 4-methyl sterol oxidase of cholesterol biosynthesis from lanosterol has been assayed to date by coupling of the NAD(P)H-dependent oxygenase to a NAD-dependent decarboxylase in a two-step incubation procedure. A simple assay of 4-methyl sterol oxidase of rat liver microsomes has now been developed with the model substrate, 4-hydroxy[ 14C]methylene-5α-cholest-7-en-3-one. In the presence of oxygen and NADPH, the 30- 14C-model substrate is oxidized directly to a 3-ketosteroid and 14CO 2, which is collected and counted. Conditions for measurement of initial rates of oxidation of the model substrate have been established. With the model substrate, the maximal velocity is four- to five-fold greater than the rate observed with 4α-methyl sterol substrates. Furthermore, when methyl sterol oxidase is measured with both the one-step and two-step assays, parallel effects are produced upon addition of either competitive or noncompetitive inhibitors in vitro; similarly, oxidative attack on each substrate is enhanced equally when rats are treated in vivo with a bile salt sequestrant. Thus, the direct one-step assay of oxidase activity with the model 4-hydroxy[ 14C]methylene sterol substrate is rapid, the observed rates are rapid, and the enzymic steps in the multienzymic, mixed function oxidase may be elucidated with this simplified procedure.