Table 2

UGT-overexpressing homogenate activities

UGT IsozymeAglycone (Km)2-aAglycone-Gluc FormationNNAL-Gluc Formation2-bNNAL-Gluc II/NNAL- Gluc I2-c
nmol/mg pmol/mg
UGT2B1Clofibric acid (12 μM)2-d 3.1  ± 0.32-e 78 ± 2.30.2
UGT1A1Bilirubin (16 μM)2.0  ± 0.3Not detected
UGT1A4Imipramine (310 μM)1.4  ± 0.2Not detected
UGT1A61-Naphthol (473 μM)94  ± 4.5Not detected
UGT1A94-Hydroxyacetophenone (73 μM)68  ± 8.2105 ± 30.6
UGT2B4Hyodeoxycholic acid (270 μM)0.23  ± 0.02Not detected
UGT2B7Androsterone (7 μM)22  ± 5.3158.5 ± 6.84.3
UGT2B154-NP (180 μM)1.3  ± 0.1Not 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.