Table 1.
Analytical conditions in the separation and quantification of glucuronides.
Figure 1.
The chemical structures of the substrates that were used in this study.
In 17α- and 17β-estradiol both the 3-OH and the 17-OH can be conjugated, mostly by different UGTs (see Figs. 2 and 3). In the case of entacapone, the glucuronidation occurs on hydroxy group in position 3.
Figure 2.
Enzyme kinetics of UGT1A7-catalyzed glucuronidation of entacapone (A) and 4-MU (B) in the absence and presence of BSA.
The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values (see Materials and Methods). The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 2.
Figure 3.
Enzyme kinetics of UGT1A8-catalyzed glucuronidation of 17β-estradiol (A), entacapone (B), 1-naphthol (C), and 4-MU (D) in the absence and presence of BSA.
The reactions with 17β-estradiol were analyzed for the formation of 17β-estradiol-3-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 2. See Materials and Methods for all further details.
Figure 4.
Enzyme kinetics of UGT1A10-catalyzed glucuronidation of 17α-estradiol (A), 17β-estradiol (B), entacapone (C), and 4-MU (D) in the absence and presence of BSA.
The reactions with 17α-estradiol and 17β-estradiol were analyzed for the formation of 17α-estradiol-3-β-D-glucuronide and 17β-estradiol-3-β-D-glucuronide, respectively. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 2. See Materials and Methods for all further details.
Figure 5.
Enzyme kinetics of UGT2A1-catalyzed glucuronidation of 6-hydroxyindole (A) and 4-MU (B), in the absence and presence of BSA.
The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 3. See Materials and Methods for all further details.
Figure 6.
Enzyme kinetics of UGT2B15-catalyzed glucuronidation of 17α-estradiol (A) and 4-MU (B), in the absence and presence of BSA.
The reaction with 17α-estradiol was analyzed for the formation of 17α-estradiol-3-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E. These values are not expression normalized because we used commercial UGT2B15 without appropriate His-tag. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 3. See Materials and Methods for all further details.
Figure 7.
Enzyme kinetics of UGT2B17-catalyzed glucuronidation of 17β-estradiol (A), 1-naphthol (B), and 4-MU (C), in the absence and presence of BSA.
The reaction with 17β-estradiol was analyzed for the formation of 17β-estradiol-17-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 3. See Materials and Methods for all further details.
Figure 8.
Enzyme kinetics of UGT2B7-catalyzed glucuronidation of 17α-estradiol (A), 17β-estradiol (B), and 4-MU (C), in the absence and presence of BSA.
The reactions with 17α-estradiol and 17β-estradiol were analyzed for the formation of 17α-estradiol-17-β-D-glucuronide and 17β-estradiol-17-β-D-glucuronide, respectively. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in Table 3. See Materials and Methods for all further details.
Table 2.
Enzyme kinetic parameters of UGTs 1A1, 1A6, 1A7, 1A8, and 1A10-catalyzed glucuronidation in the absence and presence of BSA.
Table 3.
Enzyme kinetic parameters of UGTs 2A1, 2B4, 2B7, 2B15, and 2B17-catalyzed glucuronidation in the absence and presence of BSA.