Peroxidase Activity of Liver Microsomal Vitamin D 25-Hydroxylase and Cytochrome P450 1A2 Catalyzes 25-Hydroxylation of Vitamin D3and Oxidation of Dopamine to Aminochrome

Abstract

Purified liver microsomal vitamin D 25-hydroxylase, a cytochrome P450, catalyzes 25-hydroxylation of vitamin D3in the absence of NADPH and NADPH–cytochrome P450 reductase by usingt-butyl hydroperoxide as electron donors. The rate of 25-hydroxylation was approximately the same when NADPH/NADPH–cytochrome P450 reductase ort-butyl hydroperoxide was used as electron donor. TheKmvalue for vitamin D3in the presence oft-butyl hydroperoxide was found to be 30 μM. The rates of 25-hydroxylation of 1α-hydroxyvitamin D3and 5β-cholestane-3α, 7α-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 oft-butyl hydroperoxide. Other liver microsomal cytochrome P450 forms such as taurochenodeoxycholic acid 6α-hydroxylase and cytochromes P450 1A2 and 2B4 did not catalyze 25-hydroxylation of vitamin D3in the presence of NADPH/NADPH–cytochrome P450 reductase ort-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. TheKmvalues for dopamine andt-butyl hydroperoxide were 8.6 μMand 1 mMwhen 25-hydroxylase catalyzes the formation of aminochrome in the presence oft-butyl hydroperoxide. Oxidation of dopamine to aminochrome catalyzed by the peroxidase activity of cytochrome P-450 1A2 was observed in the presence oft-butyl hydroperoxide. A linear correlation between formation of aminochrome and the amount of cytochrome P450 1A2 was found.