Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.
Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.
Postlind
H
H
Department of Pharmaceutical Biochemistry, University of Uppsala, Sweden.
Wikvall
K
K
D000818:Animals; D002762:Cholecalciferol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D007672:Kidney Cortex / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D013552:Swine
D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases