25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

J Biol Chem. 1983 Jun 10;258(11):6777-81.

Abstract

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cholecalciferol / metabolism*
  • Cholestanol
  • Cytochrome P-450 Enzyme System / metabolism*
  • Hydroxylation
  • Kinetics
  • Male
  • Microsomes, Liver / metabolism*
  • Mixed Function Oxygenases / metabolism*
  • Rats
  • Rats, Inbred Strains
  • Substrate Specificity

Substances

  • Cholecalciferol
  • Cholestanol
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases