Carrier-mediated transport of uridine diphosphoglucuronic acid across the endoplasmic reticulum membrane is a prerequisite for UDP-glucuronosyltransferase activity in rat liver.
UDP-glucuronosyltransferases (EC 2.4.1.17) is an isoenzyme family located primarily in the hepatic endoplasmic reticulum (ER) that displays latency of activity both in vitro and in vivo, as assessed respectively in microsomes and in isolated liver. The postulated luminal location of the active site of UDP-glucuronosyltransferases (UGTs) creates a permeability barrier to aglycone and UDP-GlcA access to the enzyme and implies a requirement for the transport of substrates across the ER membrane. The present study shows that the recently demonstrated carrier-mediated transport of UDP-GlcA across the ER membrane is required and rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes. We found that in both microsomes and permeabilized hepatocytes a gradual inhibition by N-ethylmaleimide (NEM) of UDP-GlcA transport into the ER produced a correspondingly increasing inhibition of 4-methylumbelliferone glucuronidation. That NEM selectively inhibited the UDP-GlcA transporter, without affecting intrinsic UGT activity, was demonstrated by showing that NEM had no effect on glucuronidation in microsomes or hepatocytes with permeabilized ER membrane. Additional evidence that UDP-GlcA transport is rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes was obtained by showing that gradual selective trans-stimulation of UDP-GlcA transport by UDP-GlcNAc, UDP-Xyl or UDP-Glc in each case produced correspondingly enhanced glucuronidation. Such stimulation of transport and glucuronidation was inhibited completely by NEM, which selectively inhibited UDP-GlcA transport.
Bossuyt
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Laboratory of Biological Chemistry, Department of Molecular Biology, Catholic University of Leuven, GHB-Herestraat 49, B-3000 Leuven, Belgium.
Blanckaert
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D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D002352:Carrier Proteins; D007527:Isoenzymes; D014535:Uridine Diphosphate Glucuronic Acid; D014537:Uridine Diphosphate N-Acetylglucosamine; D014453:Glucuronosyltransferase; D005033:Ethylmaleimide