Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase.
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 microM. At 1 microM 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 X 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.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002645:Chickens; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase