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T R Tephly

Publications and source records attributed to T R Tephly.

At least 37 records · Page 2Linked to original sources

The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268).

UGT2B7 has been cloned and expressed previously in COS cells and HK293 cells. Two forms have been identified: one with a tyrosine and one with a histidine at position 268. UGT2B7 has been shown to catalyze NSAIDs, catechol estrogens, and morphine-3- and -6-glucuronidation. cDNAs for UGT2B7Y268 and H268 were cloned and stably expressed in HK 293 cells. Studies were designed to test each form for reactivity toward a number of opioid compounds, xenobiotics such as menthol, oxazepam, and propranolol, and androgens such as androsterone and testosterone using membrane preparations derived from HK 293 cells. Both UGT2B7Y and UGT2B7H are highly reactive with many opioids, menthol, androsterone, and (R)- and (S)-propranolol, and similar kinetic values were observed. UGT2B7Y and UGT2B7H react poorly with oxazepam and no difference in (R)- or (S)-glucuronidation rate ratios was found. Thus, UGT2B7Y and H cannot account for the variability in the plasma or urine concentrations of these glucuronides in human populations. Our data suggest that UGT2B7 is a major isoform responsible for the glucuronidation of androsterone. Neither UGT2B7Y nor H catalyzes the glucuronidation of testosterone although each catalyzes the glucuronidation of epitestosterone. UGT2B7 seems to be a major human isoform responsible for the glucuronidation of opioids of the morphinan and oripavine class and is capable of catalyzing the glucuronidation of both the 3- and 6-hydroxyl moieties on these molecules. Thus, UGT2B7 plays a major role in the conversion of morphine to morphine-6-glucuronide, the potent analgesic metabolite of morphine.

Androgens↗

Glucuronidation of amines and other xenobiotics catalyzed by expressed human UDP-glucuronosyltransferase 1A3.

Glucuronide conjugation of xenobiotics containing a tertiary amine moiety represents a unique and important metabolic pathway for these compounds in humans. Previously, human UDP-glucuronosyltransferase (UGT) 1A4 was shown to be an important enzyme for the formation of quaternary ammonium-linked glucuronides. UGT1A3 is 93% identical to UGT1A4 in primary amino acid sequence. We show that human UGT1A3, transiently expressed in human embryonic kidney 293 cells, also catalyzes the N-glucuronidation of primary, secondary, and tertiary amine substrates, such as 4-aminobiphenyl, diphenylamine, and cyproheptadine. In contrast to expressed human UGT1A4, which catalyzes the glucuronidation of amines with high efficiency, glucuronidation of amines catalyzed by UGT1A3 exhibited low efficiency, suggesting that UGT1A3 makes only a limited contribution to the metabolic elimination of these compounds. The reactivity of expressed human UGT1A3 toward hydroxylated and carboxylic acid-containing compounds was also examined. In addition to amines, expressed human UGT1A3 catalyzed the glucuronidation of opioids (e.g. morphine and buprenorphine), coumarins, flavonoids (e.g. naringenin and quercetin), anthraquinones, and small phenolic compounds (e.g. 4-nitrophenol). Drugs containing a carboxylic acid moiety, such as nonsteroidal anti-inflammatory agents (e.g. naproxen and ibuprofen) and fibrates (e.g. ciprofibrate), were substrates for human UGT1A3. In contrast, compounds containing an aliphatic hydroxyl group, such as sapogenins, monoterpenoid alcohols (e.g. menthol and borneol), and androgens, were not conjugated by expressed human UGT1A3. Of the compounds tested, scopoletin, naringenin, and norbuprenorphine appeared to be the best xenobiotic substrates for human UGT1A3.

Amines↗

Glucuronidation of amine substrates by purified and expressed UDP-glucuronosyltransferase proteins.

Conjugation of many primary, secondary, and tertiary amine-containing xenobiotics with glucuronic acid can result in the formation of N-glucuronide metabolites. For carcinogenic arylamines and their N-hydroxylated metabolites, N-glucuronidation can result in the formation of either inactive metabolites or labile conjugates, which can be transported to their target tissue (urinary bladder) where they may be converted to reactive metabolites. Drugs with primary amine (e.g. dapsone) or secondary amine moieties (e.g. sulfadimethoxine and clozapine) can also be metabolized to N-glucuronides. The metabolism of a number of tertiary amine-containing pharmacological agents to quaternary ammonium-linked glucuronides represents a unique and important metabolic pathway for these compounds that is highly species-dependent. This review summarizes our present knowledge of the uridine diphosphate (UDP)-glucuronosyltransferase enzymes involved in catalyzing N-glucuronide formation. Of the more than 30 UDP-glucuronosyltransferases that have been purified or cloned and expressed, many catalyze N-glucuronide formation for primary and secondary amine substrates. In contrast, only human UDP-glucuronosyltransferases 1A3 and 1A4 have been shown to catalyze quaternary ammonium-linked glucuronide formation for aliphatic tertiary amines. The structure of the UGT1 gene complex is highly conserved across species, and it appears that a mutation in the first exon encoding UDP-glucuronosyltransferase 1A4, resulting in a pseudo-gene, may explain the inability of some species to form quaternary ammonium-linked glucuronides.

Amines↗

Immunohistochemical localization of UDP-glucuronosyltransferases in rat brain during early development.

Extrahepatic glucuronidation, such as that in the central nervous system (CNS), may play a very important role in xenobiotic disposition and may serve to protect the CNS from potentially toxic xenobiotics. UDP-glucuronosyltransferase (UGT) 1A6 is an important catalyst for phenol and polycyclic aromatic hydrocarbon glucuronidation. Studies were designed to determine the immunohistochemical localization of UGT1A6 and the steroid-reactive UGTs 2B2 and 2B3 in brain regions throughout the rat development. Neuronal cells, such as pyramidal cells, in sections from cerebral cortex and hippocampus displayed intensive UGT1A6-specific staining. UGT1A6-specific staining was also found in neuronal cells throughout the cerebral cortex, as well as in the cerebellar white matter. Glial cells revealed no apparent staining. In addition, staining for UGT1A6 was seen in choroid plexus at a later developmental stage. Although UGT1A6 staining was evident, brain sections analyzed for UGT2B2 and UGT2B3 immunoreactivity showed no significant staining. These results provide the first definitive evidence for the presence and cellular localization of UGT1A6, in neurons of developing rat brain, whereas UGT2B2 and UGT2B3 were not detected.

Animals↗

Folate and 10-formyltetrahydrofolate dehydrogenase (FDH) expression in the central nervous system of the mature rat.

10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) is a folate-binding protein that is important in folate metabolism. In addition, 10-FTHFDH catalyzes the rate-limiting step in hepatic folate-dependent formate oxidation. We measured folate concentrations and examined cellular 10-FTHFDH expression in regions of the adult rat central nervous system (CNS), to study components of CNS oxidative formate metabolism. Folate was detected in every CNS region studied; the concentrations ranged from 3 to 14% of that detected in the liver. Immunohistochemical expression of 10-FTHFDH was identified in many CNS structures. 10-FTHFDH was mostly expressed by glia, especially astrocytes and ventricular ependyma. Neuronal expression was weak but evident in the cerebral cortex, basal ganglia, cerebellum, and spinal cord. Thus, CNS tissue has the chief components of folate-dependent formate oxidation and the chief site of this oxidation appears to be glia.

Age Factors↗

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Amino Acid Sequence↗

Isolation and characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene.

Genetic polymorphisms occur in many of the drug metabolizing enzymes. However, the effect of polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases is still undescribed, despite the many reported cases of variations in glucuronidation activities. Characterization of the UGT2B15(Y85) cDNA, which was isolated from human prostate and LNCaP cell cDNA libraries, revealed 20 nucleotide differences between UGT2B15(Y85) and the previously characterized UGT2B15 protein UGT2B15(D85). However, only one of the two variations in the coding region leads to an amino acid change from aspartic acid to a tyrosine residue at position 85. The genomic DNA of 27 subjects were analysed by direct sequencing of polymerase chain reaction (PCR) products and demonstrated that UGT2B15(D85) and UGT2B15(Y85) are encoded by variant alleles prevalent in the Caucasian population. Expression of UGT2B15(D85) and UGT2B15(Y85) in HK293 cells demonstrated similar substrate specificities. Of the 65 potential substrates tested for activity, the proteins were active on phenolic compounds, coumarins, flavonoids, drugs and steroid hormones. Both proteins displayed similar Km values of 2.2 and 2.4 microM for androstane-3alpha,17beta-diol and dihydrotestosterone, respectively. However, results suggest that UGT2B15(Y85) has a higher Vmax than UGT2B15(D85). Specific reverse transcriptase (RT)-PCR analysis revealed expression of the UGT2B15 gene in a wide range of extrahepatic tissues including the human liver, kidney, testis, mammary gland, placenta, adipose, skin, uterus, prostate and lung. The wide expression of UGT2B15 in many tissues indicates that it is a major glucuronidation enzyme in humans.

Alleles↗

Human UGT2B7 catalyzes morphine glucuronidation.

A human UDP-glucuronosyltransferase (UGT) catalyzing the glucuronidation of morphine has been identified. A full length cDNA was isolated from a human liver cDNA library and found to be identical to the UGT2B7 form having a tyrosine at position 288. This cDNA was transfected into HK 293 cells, and stable expression was achieved. Cell homogenates and membrane preparations from HK 293 cells expressing UGT2B7 catalyzed the glucuronidation of morphine and other clinically significant opioid agonists, antagonists, and partial agonists. UGT2B7 catalyzed morphine glucuronidation at the 3- and 6-hydroxy positions and also mediated the formation of codeine-6-glucuronide from codeine. This represents the first demonstration of a UGT capable of catalyzing the glucuronidation of both the 3- and 6-positions of opioids. Since humans excrete morphine-3-glucuronide and morphine-6-glucuronide after morphine administration, it is likely that UGT2B7 is a major isoform in humans responsible for the metabolism of this important drug and its surrogates.

Cell Line↗

Comparison of stably expressed rat UGT1.1 and UGT2B1 in the glucuronidation of opioid compounds.

Opioids are important drugs used as analgesics, antitussives, antidiarrheals, and in the therapy of myocardial infarctions, and as antagonists of opioid intoxication. The glucuronidation of these compounds, catalyzed by UDP-glucuronosyltransferases (UGTs), is well known to be a primary step in their metabolism to hydrophilic products and in their ultimate excretion. The present study was designed to compare the reactivity and relative glucuronidation efficiencies of opioid agonists, antagonists, and partial agonists with two rat UGT isoforms; UGT1.1, which is generally considered the "bilirubin UGT," and UGT2B1, which has previously been shown to catalyze the glucuronidation of testosterone, chloramphenicol, and (-)-morphine. Rat UGT2B1, stably expressed in HK293 cells, exhibited high glucuronidation rates and catalytic efficiencies for many opioids, although values for (-)-morphine and nalorphine were the highest. In contrast, these compounds were very poor substrates for expressed rat UGT1.1. Comparably high glucuronidation rates and efficiencies were found for buprenorphine and diprenorphine with both UGT isoforms. These results suggest that opioids with morphinan-based chemical structures similar to (-)-morphine interact with UGTs differently than those with oripavine-based chemical structures similar to buprenorphine. To investigate the contribution of rat UGT1.1 and UGT2B1 in the overall rate of glucuronidation of buprenorphine in the rat liver, hepatic microsomes from Gunn rats (where UGT1.1 activity is absent) and Wistar rats (where UGT1.1 activity is present) were studied. Buprenorphine glucuronidation activity in Gunn rat liver microsomes exhibit approximately 25% of rates observed in Wistar rat liver microsomes, whereas (-)-morphine, naloxone, and naltrexone glucuronidation rates were not significantly different in microsomal preparations from Gunn and Wistar rats. These data suggest that UGT2B1 is the major hepatic enzyme involved in the glucuronidation of (-)-morphine and naloxone in livers from untreated rats, whereas buprenorphine glucuronidation is preferentially catalyzed by rat UGT1.1.

Analgesics, Opioid↗

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT).

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).

Affinity Labels↗

Glucuronidation of opioids, carboxylic acid-containing drugs, and hydroxylated xenobiotics catalyzed by expressed monkey UDP-glucuronosyltransferase 2B9 protein.

UDP-glucuronosyltransferase (UGT) 2B9, isolated from a cynomolgus monkey liver cDNA library, is 89% identical to human UGT2B7 in primary amino acid sequence, and the two expressed enzymes were previously shown to catalyze the glucuronidation of many common endogenous substrates. The purpose of the present study was to characterize the reactivity of expressed UGT2B9 with important therapeutic agents and other xenobiotics. UGT2B9, stably expressed in human embryonic kidney 293 cells, catalyzes the 3-O- and 6-O-glucuronidation of morphine and the 6-O-glucuronidation of codeine. A number of other morphinan (e.g. naloxone, naltrexone, and nalorphine) and oripavine (e.g. buprenorphine) derivatives are substrates for this enzyme. In general, morphinan derivatives are glucuronidated at higher rates, compared with oripavines; however, glucuronidation efficiency values (Vmax/KM) for the compounds are similar. Stably expressed UGT2B9 also catalyzes the glucuronidation of profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and straight-chain fatty acids at the carboxylic acid moiety. Monoterpenoid alcohols and propanolol are glucuronidated at aliphatic hydroxyl positions. Expressed UGT2B9 exhibits enantioselective glucuronidation for (R/S)-ibuprofen, (R/S)-propanolol, and (+)/(-)-menthol. The data suggest that monkey UGT2B9 and human UGT2B7 are functionally similar.

Animals↗

The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDP-glucuronosyltransferase 1.1.

Rat and human UDP-glucuronosyltransferase (UGT) 1.1 share > 70% identity in their deduced primary amino acid sequences. We have previously shown that rat UGT1.1, stably expressed in human embryonic kidney 293 cells, catalyzes the glucuronidation of bilirubin and the mixed opioid agonist/antagonist buprenorphine with high efficiency. The present study was designed to characterize the reactivity of expressed human UGT1.1 with opioid compounds and compare its substrate specificity for opioids to that of the expressed rat enzyme. The results show that both rat and human UGT1.1 catalyze the glucuronidation of opioids with a relative reactivity of buprenorphine > > nalorphine approximately naltrexone. Comparison of glucuronidation activities in livers from Crigler-Najjar type 1 patients and normal patients indicates that UGT1.1 catalyzes at least 75% of buprenorphine conjugation in normal human liver. In separate studies, the reactivity of expressed rat UGT1.1 was characterized toward various xeno-and endobiotics of various compound classes. It was found that both rat and human UGT1.1 exhibited comparable substrate specificities and efficiencies (Vmax/Km) of glucuronide formation for anthraquinones, coumarins, estrogens, flavonoids, and phenolic compounds. Neither rat nor human UGT1.1 catalyzed the glucuronidation of amines, monoterpenoid alcohols, androgens, or progestins. In general, these data indicate that rat and human UGT1.1 are functionally identical and can be considered orthologous enzymes.

Animals↗

Folate and 10-formyltetrahydrofolate dehydrogenase in human and rat retina: relation to methanol toxicity.

Ocular toxicity, a well-recognized outcome of methanol poisoning in humans, correlates with formate accumulation in blood following the ingestion of methanol. Rodents, however, are a species known to be resistant to the toxicity of formate. The present study was designed to determine whether components of folate-dependent formate oxidation, e.g., folate and 10-CHO-H4-folate dehydrogenase (10-FDH), exist in retina and whether differences in these components might explain species-determined susceptibility to methanol intoxication. Total folate levels were determined in human and rat retinal tissues and were found to be much lower than the levels in liver. However, folate levels in human retina were only 14% of those determined in rat retina. Western blot analysis was used to quantify the amount of cytosolic and mitochondrial 10-FDH in retinal tissues. Results of these studies showed that comparable amounts of this enzyme were present in both cellular compartments in each species. However, the amount of 10-FDH in human retina was approximately three times the amount found in rat retina. In order to determine the cell type(s) within the retina that contained 10-FDH, immunohistochemical staining for 10-FDH was carried out. Confocal microscopic image analysis of human and rat retinae showed colocalization of 10-FDH primarily with Muller cell protein markers [glial fibrillary acidic protein (GFAP), vimentin, and carbonic anhydrase]. Therefore, 10-FDH was found to be preferentially localized in this cell type. Since Müller cells appear to represent the target for formate-induced ocular toxicity, our data suggest that formate oxidation reactions might serve two roles, first a protective role and then a role in methanol-induced toxicity in Müller cells.

Adenosine Triphosphate↗

UDP glucuronosyltransferase in the cirrhotic rat liver.

In patients with cirrhosis, the elimination of drugs metabolized by glucuronidation is relatively preserved, in comparison with the metabolism of drugs by oxidation. This study explores this phenomenon at a molecular level. In cirrhotic rat livers the content of UDP-glucuronosyltransferase (UGT) was examined by immunohistochemistry and immunoblotting using three antibodies: (i) a polyclonal antibody directed against a broad number of UGT isoforms from both family 1 and family 2; (ii) a family 2-specific antibody; and a (iii) family 1-specific antibody. The steady state mRNA level of UGT of a family 2 isoform was also detected by northern blot analysis. The results demonstrate normal or increased UGT protein by immunohistochemistry and immunoblot in cirrhotic livers compared with controls. This was accompanied by increased steady state mRNA encoding the UGT isoform UGT2B1. In contrast, an isoform of cytochrome P450 (CYP2C11) was reduced markedly in both immunohistochemical staining and immunoblot analysis. These results suggest that in cirrhosis there is a comparative increase or at least a maintenance of UGT enzyme content and that this most likely occurs at a pretranslational level.

Animals↗

Purification and properties of two rat liver phenobarbital-inducible UDP-glucuronosyltransferases that catalyze the glucuronidation of opioids.

Glucuronidation of xenobiotics and endobiotics is catalyzed by a group of intrinsic membrane proteins of the endoplasmic reticulum of cells: the UDP-glucuronosyltransferases. Two isoforms with glucuronidation activity toward opioids have been purified and characterized from liver microsomes obtained from phenobarbital-treated Wistar rats. The proteins have been identified as the gene products of UGT2B1 and UGT1.1r. The purified proteins exhibited the same apparent KM values for morphine glucuronidation (2-3 mM). However, the purified UGT1.1r enzyme exhibited glucuronidation activity toward buprenorphine and bilirubin with high efficiency, but the UGT2B1 protein did not react with these compounds. Both purified enzymes glucuronidated chloramphenicol, 4-hydroxybiphenyl, chrysin, and ibuprofen. Flunitrazepam photoaffinity labeling was demonstrated for both enzymes, and naloxone, the opioid antagonist, antagonized the photoaffinity labeling reactions.

Amino Acid Sequence↗

Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein.

Glucuronide conjugation of tertiary amine xenobiotics represents a unique and important metabolic pathway for these compounds in humans. In this study, we show that human UDP-glucuronosyltransferase 1.4 protein, stably expressed in human embryonic kidney 293 cells, catalyzes the N-glucuronidation of primary, secondary, and tertiary amine substrates. In addition, the substrate specificity of the expressed enzyme toward many hydroxylated and carboxylic acid-containing compounds was examined. Of the hydroxylated compounds tested, only sapogenins gave glucuronidation rates comparable with those observed for amine substrates. The apparent KM and Vmax values for sapogenins were such that the efficiency of glucuronidation (Vmax/KM) for these compounds was higher than that determined for amine substrates. Human UDP-glucuronosyltranferase 1.4 also catalyzes the glucuronidation of monoterpenoid alcohols and simple phenolic compounds. The enzyme kinetic values determined for these substrates suggested that this enzyme may have relatively limited significance for the conjugation of these classes of compounds. Of the endobiotics tested, androstanediol and progestins were glucuronidated at high rates by expressed human UDP-glucuronosyltransferase 1.4 protein. The glucuronidation efficiency for 5alpha-pregnane-3beta,20alpha-diol was comparable with that determined for the sapogenins. Because UDP-glucuronosyltransferases are integral membrane proteins, the effects of different detergents on the catalytic activity of the expressed enzyme were determined. The results show that detergents (such as Lubrol PX, Emulgen 911, and Triton X-100) are inhibitory for the quaternary ammonium-linked glucuronidation of chlorpromazine and imipramine catalyzed by expressed human UDP-glucuronosyltransferase 1.4. In contrast, CHAPS and nonanoyl-N-methylglucamide are less inhibitory toward the glucuronidation of these compounds. The results suggest that human UDP-glucuronosyltransferase 1.4 may be an important enzyme for the detoxication of environmentally derived amines and sapogenins and for the conjugation of progestins.

Antipsychotic Agents↗

Cloning and expression of a rat liver phenobarbital-inducible UDP-glucuronosyltransferase (2B12) with specificity for monoterpenoid alcohols.

A full-length cDNA, HBPA2, that encodes for a new rat hepatic UDP-glucuronosyltransferase protein, designated UGT2B12, was isolated from a rat liver cDNA library. The isolated clone contains a 1590-nucleotide open reading frame flanked by 2 and 252 base pairs of 5' and 3' noncoding sequences, respectively. Human embryonic kidney 293 cells transfected with UGT2B12 expressed a protein with a subunit molecular mass of 53 kDa. The expressed protein catalyzed the glucuronidation of monoterpenoid alcohols, such as (-)-borneol, (+)-menthol, and (-)-nopol. In addition, a number of simple phenolic compounds, such as hydroxybiphenyls, 7-hydroxylated coumarins, p-nitrophenol, and food-derived substances (e.g., naringenin and eugenol), were also substrates for the expressed enzyme. Northern blot analysis showed that treatment of rats with phenobarbital increased hepatic mRNA levels for UGT2B12 approximately twofold. In addition to liver, Northern blot analysis demonstrated that UGT2B12 mRNA is present in kidney and testis.

Alcohols↗

Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin.

A chicken anti-rat polyclonal antibody to a purified rat liver UDP-glucuronosyltransferase (UGT) with catalytic activity toward opioid substrates was used to screen a liver cDNA library prepared from phenobarbital-treated Wistar rats. A number of positive clones were obtained, and one of these clones, pM1, was further characterized. Clone pM1 was found to be a full length cDNA coding for a member of the rat UGT1 gene family. Specifically, pM1 represents the full length homologue of the Gunn rat liver pseudo-gene product UGT1.1P and, therefore, has been designated UGT1.1r. The cDNA insert has an open reading frame of 1605 base pairs, which codes for a protein of 535 amino acids and is flanked by 2 and 632 base pairs of 5' and 3' noncoding sequence, respectively. The deduced amino acid sequence of pM1 contains amino acid sequences identical to the amino-terminal and internal peptides of the purified rat liver opioid UGT and to sequences reported for a rat liver bilirubin UGT [FEBS Lett. 299:183-186 (1992)]. Stable expression of UGT1.1r in human embryonic kidney 293 cells showed that a protein with a subunit molecular mass (56 kDa) identical to that of the purified protein was produced. Expressed UGT1.1r protein catalyzed the glucuronidation of buprenorphine and bilirubin at high rates. Other opioids, such as nalorphine and morphine, were also substrates for the expressed UGT1.1r protein. These results show that bilirubin and opioids can be conjugated by the same rat liver UGT.

Amino Acid Sequence↗