Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3.
A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3. The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.
Axén
E
E
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Postlind
H
H
Sjöberg
H
H
Wikvall
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D002112:Calcifediol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D017886:Durapatite; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D002112:Calcifediol