Enzyme kinetics of UGT2B15-catalyzed glucuronidation of 17α-estradiol (A) and 4-MU (B), in the absence and presence of BSA.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetics of UGT2B17-catalyzed glucuronidation of 17β-estradiol (A), 1-naphthol (B), and 4-MU (C), in the absence and presence of BSA.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetic parameters of UGTs 2A1, 2B4, 2B7, 2B15, and 2B17-catalyzed glucuronidation in the absence and presence of BSA.

<p>The glucuronidation rates are presented as expression level-normalized values ± S.E., except for the commercial UGT2B15. The 95% CI are presented in the parenthesis. The p-values were calculated using the extra sum-of-squares F-test (see <i>Materials and Methods</i> for all details).</p><p>MM, Michaelis-Menten; SI, substrate inhibition; HE, Hill equation;</p>*<p>P<0.05;</p>**<p>P<0.01;</p>***<p>P<0.001.</p

Enzyme kinetic parameters of UGTs 1A1, 1A6, 1A7, 1A8, and 1A10-catalyzed glucuronidation in the absence and presence of BSA.

<p>The glucuronidation rates are presented as expression level-normalized values ± S.E. The 95% CI are presented in the parenthesis. The p-values were calculated using the extra sum-of-squares F-test (see <i>Materials and Methods</i> for all details).</p><p>MM, Michaelis-Menten; SI, substrate inhibition; HE, Hill equation;</p>*<p>P<0.05;</p>**<p>P<0.01;</p>***<p>P<0.001.</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t002" target="_blank">Table 2</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetics of UGT2B7-catalyzed glucuronidation of 17α-estradiol (A), 17β-estradiol (B), and 4-MU (C), in the absence and presence of BSA.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

The chemical structures of the substrates that were used in this study.

<p>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.</p

Analytical conditions in the separation and quantification of glucuronides.

1<p>LOD, limit of detection; LOQ, limit of quantification; values are calculated assuming maximal injection volume;</p>2<p>The UV signal for was correlated with fluoresence for enhanced sensitivity.</p

Enzyme kinetics of UGT1A7-catalyzed glucuronidation of entacapone (A) and 4-MU (B) in the absence and presence of BSA.

<p>The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values (see <i>Materials and Methods</i>). The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t002" target="_blank">Table 2</a>.</p