Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase

• Published in: The Journal of biological chemistry Vol. 260; no. 21; pp. 11488 - 11492
• Main Authors: Paulson, S K, DeLuca, H F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.09.1985; American Society for Biochemistry and Molecular Biology
• Subjects: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kidney - enzymology; Kinetics; Malates - metabolism; Male; Mitochondria - enzymology; NADP - metabolism; Oxidoreductases; Oxygen - pharmacology; Rats; Space life sciences; Steroid Hydroxylases - analysis; Characterization; Vertebrata; Mammalia; 25-Hydroxycholecalciferol 1-monooxygenase; Rat; Enzyme; Oxidoreductase; Animal; Rodentia; Localization; Kidney
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)39055-5

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.