Assay and properties of rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase

Biochemistry. 1986 Nov 4;25(22):6821-6. doi: 10.1021/bi00370a014.


An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D3 produced from incubations of yolk sac homogenates was extracted, purified by Sephadex LH-20 chromatography and straight- and reverse-phase high-performance liquid chromatography (HPLC), and measured by a competitive binding assay using chick intestinal receptor. The reaction is linear with time for up to 45 min at a substrate concentration of 80 microM and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 X 10(-3) M) was found to inhibit 1-hydroxylation, but a 90% carbon monoxide-10% oxygen atmosphere did not, leaving open the question of involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, inhibited 1 alpha-hydroxylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase / isolation & purification
  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase / metabolism*
  • Animals
  • Calcitriol / biosynthesis
  • Chromatography, High Pressure Liquid
  • Female
  • Placenta / enzymology
  • Pregnancy
  • Rats
  • Steroid Hydroxylases / metabolism*
  • Subcellular Fractions / enzymology
  • Yolk Sac / enzymology*


  • Steroid Hydroxylases
  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase
  • Calcitriol