Investigation of the Component Reactions of Oxidative Sterol Demethylation

Microsomal enzymes of rat liver catalyze the mixed function oxidation of 4α-methyl sterols into the corresponding 4α-carboxylic acids. The 4α-carboxylic acids are further metabolized to carbon dioxide and 3-ketosteroid; the decarboxylation occurs under anaerobic conditions, and NAD+ is required. Partial purification of a NAD+-dependent microsomal enzyme that catalyzes decarboxylation of 4α-carboxylic acids has now been accomplished. Solubilization has been achieved with sodium deoxycholate, and the solubilized enzyme has been purified free of other enzymes of methyl sterol demethylase by chromatography on diethylaminoethyl-Sephadex A-50. The partially purified enzyme catalyzes decarboxylation of 3β-hydroxy-4β-methyl-5α-cholest-7-en-4α-oic acid; approximately equal amounts of CO2 and 4α-methyl-5α-cholest-7-en-3-one are formed. With the 4β-methyl-4α-carboxylic acid substrate, the enzyme exhibits a Km of 7 µm and a Vmax of 94.5 nmoles per min per mg of protein. The enzyme is selective for NAD+; with NADP+ the rate is about 5% of the net rate observed with NAD+. The pH optimum is 9.0; the enzyme is completely inactive in acidic media. Removal of bound phospholipid by treatment with either phospholipase A or C results in no loss of enzymic activity. The enzyme is not inhibited significantly by either EDTA (up to 10 mm), CN-, Fe++, GSH, Mg++, pregn-4-ene-3,20-dione, 17β-hydroxyandrost-4-en-3-one, androst-4-ene-3, 17-dione, isocitrate, or β-hydroxybutyrate. Zn++, on the other hand, inhibits at concentrations between 0.1 and 1.0 mm. Anaerobic conditions result in a 20 to 25% decrease in reaction rate. Under anaerobic conditions, addition of various electron acceptors, e.g. cytochrome c, methylene blue, or K3Fe(CN)6, did not fully restore activity.