Oxidative demethylation in sterol metabolism. Inhibition by NADH, a required cofactor

Oxidative demethylation of 4-methyl sterols by rat liver microsomes has been shown to be inhibited by low levels of NADH. Using snake venom-treated microsomes to remove endogenous NADH-utilizing activities, we observed inhibition of overall demethylation at concentrations of NADH as low as 2.5 microM. At 20 microM NADH, overall demethylation had been reduced to 60% of optimal activity. In the presence of NADH, oxidative metabolism of [30,31-14C]4,4-dimethyl-5 alpha-cholest-7-en-3 beta-ol resulted in formation of an intermediate which was metabolized anaerobically to 14CO2 in the presence of NAD+. This intermediate and its acetylated derivative had coincident mobility on silicic acid thin layer chromatograms with 3 beta-hydroxy-[14C]4 beta-methyl-5 alpha-cholest-7-en-[14C] 4 alpha-carboxylic acid sterol and its acetylated derivative. The inhibition by NADH is transitory. Normal rates return after approximately 5 min if NADH/NAD+ = 0.2, and after approximately 15 min if NADH/NAD+ = 2. At an NADH/NAD+ ratio of 10, the return to normal rates is even slower, exceeding 30 min. Further, the inhibition, though transitory, occurs again upon introduction of additional NADH to the assay mix.