Microsomal 25-hydroxylation of vitamin D2 and vitamin D3 in pig liver.
A microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D2 was purified from both male and female pigs to apparent homogeneity and a specific cytochrome P-450 content of 13 and 15.4 nmol x mg of protein-1, respectively. The enzyme also catalysed 25-hydroxylation of vitamin D3. The ratio between the 25-hydroxylase activities towards vitamin D2 and D3 was essentially the same in the different purification steps as well as in the apparently homogeneous enzyme preparation. The two enzyme activities showed the same pH optimum and decreased in parallel upon partial denaturation of the enzyme. Cholecalciferol competitively inhibited 25-hydroxylation of vitamin D2 and vice versa. The non-steroidal cytochrome P-450 inhibitor ketoconazole inhibited both enzyme activities and the Ki values were the same. The cytochrome P-450 showed the same apparent M(r), substrate specificity and N-terminal amino acid sequence as the previously purified vitamin D3 25-hydroxylase from pig liver microsomes. A monoclonal antibody raised against the vitamin D3 25-hydroxylase also recognized the vitamin D2 25-hydroxylase. The antibody immunoprecipitated the 25-hydroxylase activity towards both vitamin D2 and D3 in the purified enzyme. Taken together, the results show that the 25-hydroxylation of vitamin D2 and D3 is catalysed by the same microsomal cytochrome P-450 in pig liver microsomes. The properties of this 25-hydroxylase are discussed in relation to present knowledge concerning previously well-characterized vitamin D3 25-hydroxylases that are not able to catalyse 25-hydroxylation of vitamin D2.
Axén
E
E
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Bergman
T
T
Wikvall
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001974:Bromosuccinimide; D002762:Cholecalciferol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004872:Ergocalciferols / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017421:Sequence Analysis; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D065607:Cytochrome P-450 Enzyme Inhibitors; D004872:Ergocalciferols; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D001974:Bromosuccinimide; D007654:Ketoconazole