期刊
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
卷 90, 期 2, 页码 1210-1219出版社
ENDOCRINE SOC
DOI: 10.1210/jc.2004-0966
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资金
- NIDDK NIH HHS [DK56603-01A1] Funding Source: Medline
- NIGMS NIH HHS [GM 54995, R37 GM054995] Funding Source: Medline
Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1alpha-hydroxyvitamin D-2 (1alphaOHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1alpha-hydroxyvitamin D-3 (1alphaOHD(3)), 1alphaOHD(2), and vitamin D-2 by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D-2, 1,24(S)-dihydroxyvitamin D-2, and 1,24-dihydroxyvitamin D-3 were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1alphaOHD(2) by CYP3A4 correlated significantly with 6beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1alphaOHD(2) and 1alphaOHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120,301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D-2, 1alphaOHD(2), and 1alphaOHD(3).
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