25OHD3 4-hydroxylase activity for various recombinant P450 enzymes
Incubations were performed with 10 pmol of enzyme for 30 min. The metabolite formation rates for CYP3A4 and CYP3A5 were obtained from triplicate incubations for 10 min. The substrate 25OHD3 concentration was 12.5 μM.
| P450s | Metabolite Formation Rate | ||
|---|---|---|---|
| 24R,25(OH)2D3 | 4β,25(OH)2D3 | 4α,25(OH)2D3 | |
| pmol · min−1 · pmol−1 | |||
| CYP3A4 (b5 coexpressed) | BLQ | 2.27 ± 0.22 | 1.26 ± 0.27 |
| CYP3A5 (b5 coexpressed) | BLQ | 0.24 ± 0.03 | 0.17 ± 0.02 |
| CYP24A1a | 2.39 ± 0.63 | N.D. | N.D. |
| Other P450sb | N.D. | N.D. | N.D. |
BLQ, below limit of quantification; N.D., not detected.
↵a Metabolism of 25OHD3 by CYP24A1 was conducted in Dr. T. Sakaki's laboratory (Toyama Prefectural University). Incubation conditions: CYP24A1 (20 nM), adrenodoxin (200 nM), adrenodoxin reductase (20 nM), 1 mM NADPH, and 12.5 μM 25OHD3 in 100 mM Tris-HCl (pH 7.4, 1 mM EDTA) at 37°C for 10 min. After incubation, the products were extracted and shipped to the University of Washington. LC-MS/MS analysis was performed as described under Materials and Methods.
↵b Other P450s: CYP1A1, -1A2, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -2J2, and -3A7.