Abstract

The UDP-glucuronosyltransferases (UGTs) are a superfamily of membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to attain maximal enzyme activity. The goals of the current work were to develop a universal method to glucuronidate xenobiotic substrates using microsomes, and to apply this method to sequential oxidation-glucuronidation reactions. Three assays of UGT catalytic activity estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and morphine-3-glucuronidation, which are relatively selective probes for human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment of microsomes with the pore-forming peptide alamethicin (50 μg/mg protein) resulted in conjugation rates 2 to 3 times the rates observed with untreated microsomes. Addition of physiological concentrations of Mg²⁺ to the alamethicin-treated microsomes yielded rates that were 4 to 7 times the rates with untreated microsomes. Optimized assay conditions were found not to detrimentally affect cytochrome P450 activity as determined by effects on testosterone 6β-hydroxylation and 7-ethoxycoumarin deethylation. Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of K_m^app = 0.017 mM,V_max^app = 0.4 nmol/mg/min, and n = 1.8. Formation of acetaminophen-O-glucuronide also best fit the Hill equation, with K_m^app = 4 mM, V_max^app = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics, with K_m^app = 2 mM andV_max^app = 2.5 nmol/mg/min. Finally, alamethicin treatment of microsomes was found to be effective in facilitating the sequential oxidation-glucuronidation of 7-ethoxycoumarin.