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 sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.
Ikushiro
S
S
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1297, Japan. ikushiro@sci.himeji-tech.ac.jp
Emi
Y
Y
Kimura
S
S
Iyanagi
T
T
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013739:Testosterone / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
D009596:Nitrophenols; D014535:Uridine Diphosphate Glucuronic Acid; D013739:Testosterone; D014453:Glucuronosyltransferase; D003545:Cysteine; D005033:Ethylmaleimide; C024836:4-nitrophenol