Study of microsomal mixed function oxidative demethylation and deformylation of 4-methyl and 4-hydroxymethylene sterols.

Oxidative deformylation of 4-hydroxy[“Clmethylene-5cycholest-7-en-d-one catalyzed by rat liver microsomes has been studied using both NADH and NADPH as a source of reducing equivalents. Microsomes treated with a nonionic detergent, Triton WR-1339, will catalyze oxidative deformylation in the presence of NADPH. Both NADH and NADPH will serve as a source of reducing equivalents, however, if the microsomes are treated further with deoxycholate at a concentration of 10 mg/ml. Using deoxycholatetreated microsomes, the oxidase-catalyzing oxidative deformylation of 4-hydroxy[14Clmethylene-5cu-cholest-7-en-3-one has been compared with methyl sterol oxidase which participates in oxidative demethylation of 4,4-dimethyl-5cY-[30,3114Clcholest-7-en-3p-ol. Oxidative metabolism of both substrates was inhibited in a similar manner upon heating the microsomes at 47°C or including dithiothreitol or CNin the reaction vessel. Cytochrome c, however, caused marked inhibition (40%) of methyl sterol oxidase when NADH was used. Deformylation was virtually unaffected at concentrations of cytochrome c as high as 10 pM. Furthermore, methyl sterol oxidase was inhibited noncompetitively by 4hydroxymethylene-5a-cholest-7-en-3-one. Finally, if Tritontreated microsomes are treated with deoxycholate at a higher concentration (20 mglml) both oxidative deformylation activity and methyl sterol oxidase activity appear in the 105,000 x g supernatant fraction. However, specific activity of the oxidase catalyzing oxidative deformylation decreases significantly, whereas the specific activity of methyl sterol oxidase increases. The ratio between the two activities changes by as much as 5-fold.