25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria.
Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.
D015650:24,25-Dihydroxyvitamin D 3; D000628:Aminophylline / Q000494:pharmacology; D000818:Animals; D003577:Cytochrome P-450 Enzyme System; D004100:Dihydroxycholecalciferols / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology; D013997:Time Factors; D065668:Vitamin D3 24-Hydroxylase
D004100:Dihydroxycholecalciferols; D000628:Aminophylline; D015650:24,25-Dihydroxyvitamin D 3; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D008797:Metyrapone