Peroxidase activity of liver microsomal vitamin D 25-hydroxylase and cytochrome P450 1A2 catalyzes 25-hydroxylation of vitamin D3 and oxidation of dopamine to aminochrome.

Purified liver microsomal vitamin D 25-hydroxylase, a cytochrome P450, catalyzes 25-hydroxylation of vitamin D3 in the absence of NADPH and NADPH-cytochrome P450 reductase by using t-butyl hydroperoxide as electron donors. The rate of 25-hydroxylation was approximately the same when NADPH/ NADPH-cytochrome P450 reductase or t-butyl hydroperoxide was used as electron donor. The Km value for vitamin D3 in the presence of t-butyl hydroperoxide was found to be 30 microM. The rates of 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and 5 beta-cholestane-3 alpha, 7 alpha-diol catalyzed by 25-hydroxylase were significantly higher when the reaction proceeded in the presence of NADPH/NADPH-cytochrome P450 reductase than in the presence of t-butyl hydroperoxide. Other liver microsomal cytochrome P450 forms such as taurochenodeoxycholic acid 6 alpha-hydroxylase and cytochromes P450 1A2 and 2B4 did not catalyze 25-hydroxylation of vitamin D3 in the presence of NADPH/NADPH-cytochrome P450 reductase or t-butyl hydroperoxide. The peroxidase activity of the 25-hydroxylase also catalyzed oxidation of dopamine to aminochrome. A linear correlation between increase in aminochrome formation and increase in the amount of 25-hydroxylase was observed in the oxidation of dopamine. The Km values for dopamine and t-butyl hydroperoxide were 8.6 microM and 1 mM when 25-hydroxylase catalyzes the formation of aminochrome in the presence of t-butyl hydroperoxide. Oxidation of dopamine to aminochrome catalyzed by the peroxidase activity of cytochrome P-450 1A2 was observed in the presence of t-butyl hydroperoxide. A linear correlation between formation of aminochrome and the amount of cytochrome P450 1A2 was found.