Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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Pedersen, J.I. et al.

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.[1]

References

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria. Pedersen, J.I., Shobaki, H.H., Holmberg, I., Bergseth, S., Björkhem, I. J. Biol. Chem. (1983) [Pubmed]

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