Studies on Calciferol Metabolism

Cholecalciferol (vitamin Da) undergoes an obligatory twostep hydroxylation which is carried out sequentially first in the liver and then in the kidney, where 25-hydroxycholecalciferol-1-hydroxylase catalyzes the production of the biologically active form of vitamin Ds, 1,25-dihydroxycholecalciferol. This report describes an assay with which l-hydroxylase velocities as low as 0.2 pmole per mm can be measured. The level of enzyme activity varies over a 6-fold range depending whether the mitochondria are isolated from cholecalciferol-deficient ( -D3) (V,,, = 12 to 16 pmoles per min per mg of protein) or -repleted ( +D3) (V,,, = 2 to 3 pmoles per min per mg of protein) chicks; the K, for 25-hydroxycholecalciferol was 1 to 2 x 10m7 in both mitochondrial preparations. Succinate and malate are the most effective electron donors for the hydroxylation. The pattern of inhibition of l-hydroxylase activity seen in the presence of antimycin, rotenone, oligomycin, and CN when succinate or malate were electron donors is consistent with the concept that the l-hydroxylase is similar in nature to many other mixed function oxidases. The partition coefficient, K, for CO inhibition of the 1-hydroxylase was found to be 1.0, a value comparable to that for other steroidogenic hydroxylases. The wavelength of light most effective in reversing CO inhibition was 450 nm. This is the first direct evidence of the involvement of cytochrome P-450 with the renal I-hydroxylase. The presence of Ca*+, 10m5 M, or phosphate, 5 X 10T3 M, both effected a 50% inhibition of the I-hydroxylase. This is consistent with the view that changes in the mitochondrial fluxes of these ions may be important in the regulation of the production of the hormonally active form of vitamin D. Also 2 X 10e6 M Mn*+, 1 x 10m5 M Sr2+ effected a 50 % inhibition of the I-hydroxylase, but 0.1 M Ba2+, 0.4 mM ethane diphosphonate, 10 mM acetate, or 10 mrd sulfate had no effect on enzyme activity. The variation in renal 25-hydroxycholecalciferol-l-hydroxylase activity with cholecalciferol status and the inhibi-