Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.
1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.
Inaba
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M
Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences 53706.
Burgos-Trinidad
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M
DeLuca
H F
HF
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D002117:Calcitriol / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D004790:Enzyme Induction; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D010504:Periodic Acid / Q000494:pharmacology; D015203:Reproducibility of Results; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D065668:Vitamin D3 24-Hydroxylase
D010504:Periodic Acid; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; C009288:metaperiodate; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate