Assay and properties of rat yolk sac 25-hydroxyvitamin D/sub 3/ 1. cap alpha. -hydroxylase

Journal Article · Tue Nov 04 04:00:00 EST 1986 · Biochemistry; (United States)

OSTI ID:6439605

Paulson, S K; Phelps, M; DeLuca, H F

An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D/sub 3/ 1..cap alpha..-hydroxylase (1..cap alpha..-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D/sub 3/ 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 ..mu..M and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 x 10/sup -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..cap alpha..-hydroxylation.

• Research Organization: Univ. of Wisconsin, Madison
• OSTI ID: 6439605
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 25:22; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOCHEMISTRY
CARBON COMPOUNDS
CARBON MONOXIDE
CARBON OXIDES
CELL CONSTITUENTS
CHALCOGENIDES
CHEMISTRY
CHOLECALCIFEROL
CHROMATOGRAPHY
CYTOCHROMES
DISTRIBUTION
EGGS
ELEMENTS
ENZYMES
FETAL MEMBRANES
HYDROXYLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANES
MICROSOMES
NONMETALS
ORGANIC COMPOUNDS
ORGANOIDS
OXIDES
OXIDOREDUCTASES
OXYGEN
OXYGEN COMPOUNDS
PIGMENTS
PROTEINS
RADIORECEPTOR ASSAY
RATS
RODENTS
SEPARATION PROCESSES
SUBCELLULAR DISTRIBUTION
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
VITAMIN D
VITAMINS