Mixed function oxidases in sterol metabolism. Source of reducing equivalents.

Either NADH or NADPH can serve as a cofactor for oxidative demethylation of [30,31-14C]4,4-dimethyl-5 alpha-cholest-7-en-3 beta-ol and oxidative deformylation of 4-hydroxy[14C]methylene-5 alpha-cholest-7-en-3-one. This report suggests that the cofactors interact with these two oxidase systems differently depending upon whether the reduced cofactor arises intra- or extramicrosomally. Marked differences in oxidative activity are observed depending on whether NADPH is generated in the microsomes or is added as an exogenous cofactor. Thus, the concentration of added NADPH required to yield maximal rates of sterol oxidation is 500 muM or greater. Nearly equivalent rates of sterol oxidation are obtained from NADPH generated in the microsomes where the NADPH concentration is no greater than 0.454 muM. Similar results are observed with NADH. In this case, NADH is generated in the microsomes from added NAD+ by microsomal reactions. The rate of sterol oxidation when NADH is generated from added NAD+ is nearly the same as that obtained from added NADH; although the concentration of NADH generated from NAD+ is 0.403 muM, the concentration of added NADH is 100 muM, and Km for added NADH is 1.7 muM.

Mixed function oxidases in sterol metabolism. Separate routes for electron transfer from NADH and NADPH.

Oxidative deformylation of 4-hydroxy[14C]methylene-5alpha-cholest-7-en-3-one and oxidative demethylation of [30,31-14C]4,4-dimethyl-5alpha-cholest-7-en-3beta-ol by rat liver microsomes have been compared with regard to the manner in which electrons are introduced from both NADH and NADPH. Evidence suggests that NADH and NADPH support oxidation of both substrates via separate routes of electron transfer. Thus, 10 micron cytochrome c will inhibit NADPH-supported oxidation to 40 to 50% of control activity leaving NADH-supported oxidation unaffected. Also, treatment of microsomes with subtilisin diminishes NADPH-supported oxidation to 10 to 30% of control activity for either substrate to 70 to 90% of control activity while NADH-supported oxidative activity is virtually unaffected. Studies on the oxidase activities and NADPH-cytochrome c reductase as well as NADH-ferricyanide reductase have shown marked differences in activity in the presence of inhibitors. Thus, 9 mM 2'-AMP inhibits NADPH-cytochrome c reductase to 10 to 20% of control activity while NADPH-supported oxidative demethyl ation and deformylation are essentially unchanged. Mersalyl at 15 to 25 nmol/mg of microsomal protein inhibits both reductases to 20 to 40% of control activity; oxidative demethylation is unaffected and oxidative deformylation stimulated slightly when NADPH is used. Finally, antibody to NADPH-cytochrome c reductase inhibits oxidase activity for either substrate to 70 to 90% of control activity while reductase activity is inhibited to 10 to 30% of control activity.