title: Assay and properties of rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase.
abstract: 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.
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002117:Calcitriol / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D005260:Female; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D015017:Yolk Sac / Q000201:enzymology*
chemical_list: D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol