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Measurement and characterization of C-3 epimerization activity toward vitamin D3

2005; Elsevier BV; Volume: 436; Issue: 1 Linguagem: Inglês

10.1016/j.abb.2005.01.017

1096-0384

Maya Kamao, Susumi Hatakeyama, Toshiyuki Sakaki, Natsumi Sawada, Kuniyo Inouye, Noboru Kubodera, G. Satyanarayana Reddy, Toshio Okano,

Pharmacogenetics and Drug Metabolism

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 Mg2+. 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 (Vmax) and the Michaelis constant (Km), 25-hydroxyvitamin D3 [25(OH)D3] exhibited the highest specificity for the epimerization at C-3 among 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], 25(OH)D3, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], 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(α → β)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D3 at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.