Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydr yl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA ) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inac tivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-Glc UA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependen t stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of th e microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide a ntibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immun opurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA p rotected against the labeling as it did with the NEM-sensitive inactivation These data suggest the involvement of a sulfhydryl residue of microsomal p rotein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulatio n of UDP-GlcUA uptake into microsomal vesicles. (C) 1999 Elsevier Science B .V. All rights reserved.