Uptake Of UDP-glucuronic Acid Research Articles

Transporter-mediated uptake of UDP-glucuronic acid by human liver microsomes: assay conditions, kinetics, and inhibition.

This study characterized the kinetics, variability, and factors that affect UDP-glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the high- and low-affinity components differed by more than an order of magnitude (13 ± 6 vs. 374 ± 175 µM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 ± 26 vs. 131 ± 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 μM) and high (1000 μM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the high- and low- affinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 μM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds.

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.

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.

Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.

The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.

Hepatic regeneration is associated with preservation of microsomal glucuronidation

Significant controversy exists regarding the regulation of glucuronidation during the process of hepatic regeneration. We used a partial hepatectomy rat model to elucidate the effects of hepatic regeneration on the various components of the microsomal glucuronidation system. Hepatic microsomes were prepared by standard sucrose density centrifugation, coupled with a modified technique involving Percoll centrifugation. Microsomal uridine diphosphate (UDP)-glucuronosyltransferase (UGT) protein expression and UGT messenger RNA (mRNA) levels were measured by Western and Northern blotting. UGT enzyme activity was determined toward two prototypical aglycones, p-nitrophenol and estrone, in intact and digitonin-treated microsomes. Microsomal uptake of the cosubstrate for all glucuronidation reactions, UDP-glucuronic acid (UDP-GlcUA), was determined using a rapid-filtration assay. Microsomal enrichment after hepatectomy was preserved only when the Percoll method was used. Microsomal UGT protein expression and UGT mRNA levels were unaltered after hepatectomy. UGT enzyme activity toward estrone was unchanged 1 day posthepatectomy compared with sham laparotomy controls. Similarly, p-nitrophenol glucuronide formation was unaffected by hepatic regeneration 1, 2, and 5 days posthepatectomy when digitonin-treated microsomes were used. Glucuronidation of p-nitrophenol in intact microsomes was increased in partial hepatectomy compared with sham-operated controls at 1 and 2 days. This increase was not attributable to changes in microsomal UDP-GlcUA uptake, which was comparable in both groups. We conclude that microsomal glucuronidation, in contrast to other well characterized hepatic metabolic functions, is highly preserved during liver regeneration. (Hepatology 1996 Nov;24(5):1250-5)

Hepatic regeneration is associated with preservation of microsomal glucuronidation.

Significant controversy exists regarding the regulation of glucuronidation during the process of hepatic regeneration. We used a partial hepatectomy rat model to elucidate the effects of hepatic regeneration on the various components of the microsomal glucuronidation system. Hepatic microsomes were prepared by standard sucrose density centrifugation, coupled with a modified technique involving Percoll centrifugation. Microsomal uridine diphosphate (UDP)-glucuronosyltransferase (UGT) protein expression and UGT messenger RNA (mRNA) levels were measured by Western and Northern blotting. UGT enzyme activity was determined toward two prototypical aglycones, p-nitrophenol and estrone, in intact and digitonin-treated microsomes. Microsomal uptake of the cosubstrate for all glucuronidation reactions, UDP-glucuronic acid (UDP-GlcUA), was determined using a rapid-filtration assay. Microsomal enrichment after hepatectomy was preserved only when the Percoll method was used. Microsomal UGT protein expression and UGT mRNA levels were unaltered after hepatectomy. UGT enzyme activity toward estrone was unchanged 1 day posthepatectomy compared with sham laparotomy controls. Similarly, p-nitrophenol glucuronide formation was unaffected by hepatic regeneration 1, 2, and 5 days posthepatectomy when digitonin-treated microsomes were used. Glucuronidation of p-nitrophenol in intact microsomes was increased in partial hepatectomy compared with sham-operated controls at 1 and 2 days. This increase was not attributable to changes in microsomal UDP-GlcUA uptake, which was comparable in both groups. We conclude that microsomal glucuronidation, in contrast to other well characterized hepatic metabolic functions, is highly preserved during liver regeneration.

Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.