Chick kidney microsomal cytochrome P-450 involvement in the metabolism of 25-hydroxyvitamin D 3

Abstract

A new metabolic activity is described for liver, kidney, and intestinal mucosal microsomes of rachitic chicks that is associated with the metabolism of 25-hydroxyvitamin D 3 (25-OH-D 3). Cytochrome P-450 was shown to be an integral component of this metabolism, supporting the monooxygenase nature of the microsomal activities. The 25-OH-D 3 was the specific substrate inasmuch as the reactions could not be demonstrated to occur when vitamin D 3, 1,25-dihydroxyvitamin D 3, or cholesterol was the substrate. The microsomal cytochrome P-450 concentrations of rachitic chick liver, kidney, and intestinal mucosa were 0.108, 0.068, and 0.270 nmol/mg of protein, respectively. The cytochrome b 5 concentrations of the tissues in the same order were 0.198, 0.123, and 0.306 nmol/mg of protein. Normal chick tissue P-450 and b 5 concentrations were the same as those in the rachitic. The kidney enzyme has a pH optimum of 6.6 to 7.5 and an apparent K m of 2.5 μ m. At a substrate concentration of 0.417 μ m, the rate of 25-OH-D 3 metabolism was proportional to a microsomal protein concentration of up to 3 mg/ml. The activity was inhibited 100% by 10 μ m n-ethylmaleimide, 1 m m o-phenanthroline, 1 m m metyrapone, or by a carbon monoxide:oxygen mixture of 90:10 ( v v ). A 50% inhibition of activity was produced by 5 m m sodium cyanide, but 10 μ m antimycin A and rotenone were ineffective. Supporting evidence for the participation of a cytochrome P-450 species in the renal microsomal metabolism of 25-OH-D 3 included the type I substrate-induced spectral shift at 0.5 μ m 25-OH-D 3 with a modified positive absorption at 405 nm and a negative absorption at 422 nm. These findings suggest that a metabolically active cytochrome P-450-substrate complex is involved in the renal microsomal biotransformations of 25-OH-D 3 as a component of the regulatory processes of vitamin D 3 metabolism.