UGT-overexpressing homogenate activities
| UGT Isozyme | Aglycone (Km)2-a | Aglycone-Gluc Formation | NNAL-Gluc Formation2-b | NNAL-Gluc II/NNAL- Gluc I2-c |
|---|---|---|---|---|
| nmol/mg | pmol/mg | |||
| UGT2B1 | Clofibric acid (12 μM)2-d | 3.1 ± 0.32-e | 78 ± 2.3 | 0.2 |
| UGT1A1 | Bilirubin (16 μM) | 2.0 ± 0.3 | Not detected | |
| UGT1A4 | Imipramine (310 μM) | 1.4 ± 0.2 | Not detected | |
| UGT1A6 | 1-Naphthol (473 μM) | 94 ± 4.5 | Not detected | |
| UGT1A9 | 4-Hydroxyacetophenone (73 μM) | 68 ± 8.2 | 105 ± 3 | 0.6 |
| UGT2B4 | Hyodeoxycholic acid (270 μM) | 0.23 ± 0.02 | Not detected | |
| UGT2B7 | Androsterone (7 μM) | 22 ± 5.3 | 158.5 ± 6.8 | 4.3 |
| UGT2B15 | 4-NP (180 μM) | 1.3 ± 0.1 | Not detected |
↵2-a Aglycone glucuronidation assays were performed using UGT-overexpressing cell homogenates (250 μg of protein) as described under Materials and Methods. The aglycone concentrations used were 10 times higher than the published Km values for each UGT isozyme, with all incubations performed for 2 h.
↵2-b NNAL glucuronidation assays were performed using 5 mg of homogenate protein for UGT2B7-overexpressing cells, 2.5 mg for UGT1A9, 0.25 mg for UGT2B1, and 8 mg homogenate protein for all other UGT-overexpressing cell lines, and 1 mM (UGT2B1) or 5 mM (all other UGTs) NNAL, for 5 min (UGT2B1) or 30 min (all other UGTs).
↵2-c Ratio of NNAL-Gluc II to NNAL-Gluc I in NNAL glucuronidation assays using homogenates from UGT-overexpressing cell lines.
↵2-d The published Kmvalues of UGT isozymes for the corresponding aglycone are as follows: UGT2B1/clofibric acid, Pritchard et al., 1994; UGT1A1/bilirubin,Coffman et al., 1995; UGT1A4/imipramine, Green et al., 1995; UGT1A6/1-naphthol, Mackenzie et al., 1993; UGT1A9/4-hydroxyacetophenone, Ebner and Burchell, 1993; UGT2B4/hyodeoxycholic acid, Abid et al., 1997; UGT2B7/androsterone,Pillot et al., 1993; UGT2B15/4-NP, Green et al., 1994.
↵2-e Mean ± S.D. of triplicate results.