Enzyme kinetic analysis of carbazeran 4-oxidation and O6-benzylguanine 8-oxidation in enzymatic incubations containing human liver microsomes, cytosol, or S9 fraction
Vmax, apparent Km, fraction unbound (fu), corrected Km, and unbound intrinsic clearance (Clint,u) were calculated as described under Materials and Methods. Data are expressed as mean ± S.E.M. of three independent experiments conducted in duplicate or triplicate.
| Sample | Vmax | Vmax | Apparent Km | Fraction Unbounda | Corrected Km | Clint,u | Clint,u |
|---|---|---|---|---|---|---|---|
| pmol/min per milligram of protein | pmol/min per picomole of AOX-1 | μM | fu | μM | μl/min per milligram of protein | μl/min per picomole of AOX-1 | |
| Carbazeran 4-oxidation | |||||||
| Human liver microsomes (without NADPH)b | 187 ± 17 | 14.5 ± 1.3 | 7.2 ± 1.0 | 0.87 ± 0.01 | 6.3 ± 0.9 | 30 ± 1 | 2.32 ± 0.11 |
| Human liver microsomes (with NADPH)b | 179 ± 14 | 13.8 ± 1.0 | 7.1 ± 0.5 | 0.87 ± 0.01 | 6.2 ± 0.4 | 29 ± 1 | 2.21 ± 0.04 |
| Human liver cytosol | 1290 ± 138 | 20.2 ± 2.2 | 6.3 ± 0.7 | 0.94 ± 0.02 | 5.9 ± 0.6 | 217 ± 5 | 3.41 ± 0.08 |
| Human liver S9 fraction (without NADPH) | 671 ± 83 | 12.6 ± 1.6 | 4.9 ± 0.3 | 0.96 ± 0.01 | 4.7 ± 0.3 | 144 ± 19 | 2.61 ± 0.34 |
| Human liver S9 fraction (with NADPH) | 682 ± 83 | 12.8 ± 1.6 | 4.9 ± 0.3 | 0.96 ± 0.01 | 4.7 ± 0.3 | 144 ± 10 | 2.61 ± 0.17 |
| O6-Benzylguanine 8-oxidation | |||||||
| Human liver microsomes (without NADPH)b | N/Ac | N/Ac | N/Ac | 0.99 ± 0.03 | N/Ac | N/Ac | N/Ac |
| Human liver microsomes (with NADPH)b | 94 ± 11 | 7.2 ± 0.9 | 43 ± 4 | 0.99 ± 0.03 | 42 ± 4 | 2.3 ± 0.3 | 0.17 ± 0.03 |
| Human liver cytosol | 1254 ± 102 | 19.7 ± 1.6 | 70 ± 8 | 1.01 ± 0.02 | 71 ± 8 | 18 ± 2 | 0.28 ± 0.03 |
| Human liver S9 fraction (without NADPH) | 445 ± 10 | 8.35 ± 0.19 | 94 ± 3 | 0.96 ± 0.01 | 90 ± 3 | 4.9 ± 0.2 | 0.09 ± 0.003 |
| Human liver S9 fraction (with NADPH) | 454 ± 5 | 8.52 ± 0.09 | 85 ± 9 | 0.96 ± 0.01 | 81 ± 8 | 5.7 ± 0.6 | 0.10 ± 0.01 |
↵a fu from 10 μM carbazeran and 200 μM O6-benzylguanine in the respective enzymes (Supplemental Table S3).
↵b Human liver microsomes from Pool 2.
↵c The V vs. [S] plot did not reach plateau and kinetic values could not be determined. Owing to solubility limit, the maximum substrate concentration was 100 μM.