Table 1.
Effect of ANF (25 μM) on testosterone hydroxylase activities of P450 3A4 wild-type and site-directed mutant enzymes using 25 μM testosterone as substrate
| 3A4 Sample | MeOH*
|
ANF*
|
Fold stimulation by ANF
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| 2β-OH | 6β-OH | 15β-OH | 2β-OH | 6β-OH | 15β-OH | 2β-OH | 6β-OH | 15β-OH | |
| WT | 0.4 (10) | 3.4 (85) | 0.2 (5) | 1.1 (14) | 6.0 (78) | 0.6 (8) | 2.8 | 1.8 | 3.0 |
| L211F | 0.8 (11) | 5.9 (83) | 0.4 (6) | 1.5 (15) | 7.2 (75) | 1.0 (10) | 1.9 | 1.2 | 2.5 |
| D214E | 1.1 (14) | 5.9 (76) | 0.8 (10) | 1.8 (16) | 8.4 (73) | 1.2 (11) | 1.6 | 1.4 | 1.5 |
| L211F/D214E | 1.1 (14) | 6.3 (79) | 0.6 (7) | 1.0 (12) | 6.7 (80) | 0.73 (8) | 0.9 | 1.1 | 1.2 |
*
Values are expressed as nanomoles of product formed per minute per nanomole of P450 and are the average of duplicate determinations. Numbers in parentheses represent the rate of metabolite formation as a percentage of the total (2β-OH + 6β-OH + 15β-OH testosterone).