Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase.
The subcellular location and some properties of the rat kidney 25-hydroxyvitamin D3-1 alpha-hydroxylase are described. Enzyme activity can be measured as previously discussed (Tanaka, Y., and DeLuca, H.F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 196-199) using saturating substrate (25-hydroxyvitamin D3) concentrations. The reaction is linear with time for up to 30 min at a substrate concentration of 80 microM and 9-11 mg/ml mitochondrial protein. The enzyme, located in the mitochondria, requires molecular oxygen and a source of NADPH. Succinate supplies NADPH for 1 alpha-hydroxylation through reversal of electron transport and transhydrogenation as shown by inhibition with antimycin A and dinitrophenol. Malate supplies NADPH for the reaction via the mitochondrial malic enzyme or malate dehydrogenase and transhydrogenase as indicated by the lack of inhibition by antimycin A but inhibition with dinitrophenol. Metyrapone and carbon monoxide both inhibit 1 alpha-hydroxylation indicating the involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, has no effect on 1 alpha-hydroxylation.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000032:analysis*; D000818:Animals; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D051381:Rats; D013250:Steroid Hydroxylases / Q000032:analysis*
D008293:Malates; D009249:NADP; C030298:malic acid; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D010100:Oxygen