A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase.
A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.
Brown
A J
AJ
Department of Biochemistry, University of Wisconsin, College of Agricultural and Life Sciences, Madison 53706.
Perlman
K
K
Schnoes
H K
HK
DeLuca
H F
HF
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000032:analysis*; D000818:Animals; D002112:Calcifediol / Q000138:chemical synthesis; D002645:Chickens; D007668:Kidney / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000032:analysis*; D014316:Tritium
D014316:Tritium; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002112:Calcifediol