A functional role for histidyl residues of the UDP-glucuronic acid carrier in rat liver endoplasmic reticulum membranes.
Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.
Battaglia
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Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Radominska-Pandya
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A
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001557:Benzenesulfonates / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006639:Histidine / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009574:Nitro Compounds / Q000494:pharmacology; D019920:Photoaffinity Labels; D051381:Rats; D017207:Rats, Sprague-Dawley; D014535:Uridine Diphosphate Glucuronic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000494:pharmacology
D000098:Acetophenones; D001557:Benzenesulfonates; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D009574:Nitro Compounds; D019920:Photoaffinity Labels; D014539:Uridine Diphosphate Sugars; C067691:2,4-difluoro-5-nitrobenzene sulfonic acid; D014535:Uridine Diphosphate Glucuronic Acid; D006639:Histidine; D004047:Diethyl Pyrocarbonate; C006821:4-bromophenacyl bromide