Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase

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Abstract
Chick kidney mitochondrial 25-hydroxyvitamin D3 24-hydroxylase has been solubilized with sodium cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for maximal 24-hydroxylase activity. The product 24 (R), 25-dihydroxyvitamin D3 was identified by cochromatography with synthetic compound on straight-phase and reversed-phase high-performance liquid chromatography and by periodate oxidation. The enzyme has an apparent Km for 25-hydroxyvitamin D3 of 0.67 .mu.M. At 1 .mu.M 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 production is linear with time for up to 15 min and with protein concentrations of up to 2 mg/mL. The antioxidant diphenyl-p-phenylenediamine (1.3 .times. 10-4 M) has no effect on this reaction. Reconstituted 24-hydroxylase activity is enhanced by the addition of NaCl and KCl up to 100 mM, with higher concentrations having an inhibitory effect. 1.alpha.-Hydroxylase is not present in this preparation from vitamin D replete chicks. The similarities of this reconstituted system to the 25-hydroxyvitamin D3 1.alpha.-hydroxylase and the adrenal systems suggest that the 25-hydroxyvitamin D3 24-hydroxylase is also a cytochrome P-450 type mixed-function oxidase.