25-hydroxyvitamin D hydroxylation. Evidence for a dioxygenase activity of solubilized renal mitochondrial cytochrome P-450.
Upon solubilization and partial purification, renal mitochondrial P-450 catalyzes both the 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in the absence of NADPH. Neither 1 alpha,25-dihydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 is further metabolized by the enzyme. Under similar conditions, P-450 obtained from hepatic microsomes or adrenal mitochondria is inactive as a 25-hydroxyvitamin D3 hydroxylase. Both hydroxylations are heat-sensitive and are inhibited by 1 alpha,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin, 25,26-dihydroxyvitamin D3, EDTA, menadione, dithiothreitol, and cadmium but not by carbon monoxide. Cumene hydroperoxide does not facilitate either hydroxylation and no lipid peroxides can be detected in the system either prior to or after incubation with the substrate. Based on these data, it is proposed that during these hydroxylations P-450 is acting as a dioxygenase. The first step in the sequence of reactions is postulated to be a P-450-catalyzed formation of a hydroperoxide at carbon 1 of a substrate molecule. This is followed by the 25-hydroxyvitamin D hydroperoxide-supported, P-450-catalyzed hydroxylation of another molecule of substrate at either the 1 alpha or the 24 position. During the cycle 1 alpha,25-dihydroxyvitamin D3 is also generated from the hydroperoxide. From this mechanism, it can be concluded that one form of P-450 is capable of catalyzing the production of both 1 alpha,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D000818:Animals; D002112:Calcifediol / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006900:Hydroxylation; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity; D065668:Vitamin D3 24-Hydroxylase; D014812:Vitamin K / Q000494:pharmacology
D014812:Vitamin K; D003577:Cytochrome P-450 Enzyme System; D006735:Horseradish Peroxidase; D002374:Catalase; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002112:Calcifediol; D004229:Dithiothreitol