Inhibition and active sites of UDP-glucuronosyltransferases 2B7 and 1A1.

Two human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A1, catalyze the glucuronidation of many endo- and xenobiotics. Although UGT1A1 uniquely catalyzes the glucuronidation of the endobiotic, bilirubin, and UGT2B7 uniquely catalyzes the glucuronidation of morphine to both the 3-0 glucuronide and the 6-0 glucuronide, both catalyze the glucuronidation of the mixed opioid agonist/antagonist buprenorphine with high efficiency. Etonitazenyl, a mu opioid receptor antagonist, was found to inhibit competitively opioid, steroid, and other substrate glucuronidation reactions catalyzed by UGT2B7. Data showing several benzodiazepines and alternative substrates interacting competitively support previous work, which indicates a single binding domain within UGT2B7. Etonitazenyl also competitively inhibited the glucuronidation of buprenorphine catalyzed by UGT1A1. However, neither etonitazenyl nor buprenorphine inhibited bilirubin glucuronidation except at very high concentrations. Therefore, it is unlikely that buprenorphine therapy for opioid or other drug addiction would influence bilirubin glucuronidation and lead to hyperbilirubenmia. Anthraflavic acid and catechol estrogen glucuronidation, catalyzed by UGT1A1, was also not inhibited by etonitazenyl or buprenorphine. Reactions catalyzed by UGT1A6 were not affected by etonitazenyl. These studies indicate that UGT2B7 has one binding site and that UGT1A1 has two or more binding sites for xenobiotics and endobiotics.

Measurements of UDP- glucuronosyltransferase (UGT) activities.

Mammalian UDP-glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep-Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP-glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP-glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.