Measurement and characterization of C-3 epimerization activity toward vitamin D3.
Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.
Kamao
Maya
M
Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe 658-8558, Japan.
Hatakeyama
Susumi
S
Sakaki
Toshiyuki
T
Sawada
Natsumi
N
Inouye
Kuniyo
K
Kubodera
Noboru
N
Reddy
G Satyanarayana
GS
Okano
Toshio
T
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism; D002413:Cations, Divalent; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000031:analogs & derivatives / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology; D019998:Racemases and Epimerases / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D002413:Cations, Divalent; D002762:Cholecalciferol; D009249:NADP; D019298:Glucose-6-Phosphate; D003577:Cytochrome P-450 Enzyme System; D005954:Glucosephosphate Dehydrogenase; D019998:Racemases and Epimerases; D002117:Calcitriol; D008274:Magnesium; C051883:maxacalcitol