Purification and properties of rabbit liver estrone and p-nitrophenol UDP-glucuronyltransferases

doi:10.1016/0003-9861(81)90314-3

Archives of Biochemistry and Biophysics

Volume 209, Issue 2, July 1981, Pages 565-578

https://doi.org/10.1016/0003-9861(81)90314-3 Get rights and content

Abstract

Estrone and p-nitrophenol UDP-glucuronyltransferases from rabbit liver microsomes have been separated and purified to homogeneity by DEAE-cellulose chromatography and affinity chromatography on UDP-hexanolamine Sepharose-4B. Both enzyme preparations exhibited subunit molecular weights of 57,000. Estrone and p-nitrophenol UDP-glucuronyltransferases appear to exist as tetramers with an apparent molecular weight of 230,000 as determined by gel chromatography. Estrone UDP-glucuronyltransferase was completely inactive unless assayed in the presence of phosphatidylcholine. In the absence of phospholipid, considerable p-nitrophenol UDP-glucuronyltransferase enzyme activity could be detected; however, a fourfold increase in specific activity occurred in the presence of phospholipid. Estrone UDP-glucuronyltransferase could not catalyze the glucuronidation of p-nitrophenol, whereas the p-nitrophenol UDP-glucuronyltransferase displayed slight activity toward estrone. Characterization of these enzymes revealed that estrone and p-nitrophenol UDP-glucuronyltransferases are clearly different proteins. Both enzymes demonstrated charge heterogeneity on polyacrylamide isoelectric focusing, with estrone and p-nitrophenol UDP-glucuronyltransferases exhibiting isoelectric points of 7.6 and 6.8, respectively. Amino acid analysis of the purified enzymes demonstrated that estrone UDP-glucuronyltransferase contained 60% hydrophobic amino acids while p-nitrophenol UDP-glucuronyltransferase contained 53% hydrophobic amino acids. Limited proteolysis in the presence of sodium dodecyl sulfate resulted in clear differences in the peptide map composition of the respective transferase. Also, each enzyme exhibited a different pH optima for the expression of maximal catalytic activity.

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The UGTome: The expanding diversity of UDP glycosyltransferases and its impact on small molecule metabolism
2019, Pharmacology and Therapeutics
Citation Excerpt :
Tukey and Tephly purified UGT proteins that exhibited either estrone or p-nitrophenol glucuronidation activities from rabbit liver. These forms showed apparent molecular weights of 230 kDa based on gel filtration (Tukey & Tephly, 1981). Given that the proteins were found by SDS-PAGE to have monomeric molecular weights of ∼53 kDa (consistent with later molecular cloning studies), the data suggested that UGTs might form tetramers.
The UDP glycosyltransferase (UGT) superfamily of enzymes is responsible for the metabolism and clearance of thousands of lipophilic chemicals including drugs, toxins and endogenous signaling molecules. They provide a protective interface between the organism and its chemical-rich environment, as well as controlling critical signaling pathways to maintain healthy tissue function. UGTs are associated with drug responses and interactions, as well as a wide range of diseases including cancer. The human genome contains 22 UGT genes; however as befitting their exceptionally diverse substrate ranges and biological activities, the output of these UGT genes is functionally diversified by multiple processes including alternative splicing, post-translational modification, homo- and hetero-oligomerization, and interactions with other proteins. All UGT genes are subject to extensive alternative splicing generating variant/truncated UGT proteins with altered functions including the capacity to dominantly modulate/inhibit cognate full-length forms. Heterotypic oligomerization of different UGTs can alter kinetic properties relative to monotypic complexes, and potentially produce novel substrate specificities. Moreover, the recently profiled interactions of UGTs with non-UGT proteins may facilitate coordination between different metabolic processes, as well as providing opportunities for UGTs to engage in novel ‘moonlighting’ functions. Herein we provide a detailed and comprehensive review of all known modes of UGT functional diversification and propose a UGTome model to describe the resulting expansion of metabolic capacity and its potential to modulate drug/xenobiotic responses and cell behaviours in normal and disease contexts.
Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions
2009, Biochemical Pharmacology
UDP-glucuronosyltransferases (UGTs) represent major Phase II enzymes involved in detoxification of endo- and xenobiotics, including many drugs. The intraluminal orientation of the active site of UGTs in endoplasmic reticulum membranes necessitates a number of transporters in these membranes, for example, for UDP-glucuronic acid and glucuronides, the latter being insufficiently characterized. In addition, accumulating evidence suggests that UGTs are functional as homo- and heterodimers in monoglucuronide formation. They may form tetramers in diglucuronide formation. UGT oligomers probably serve to stabilize UGT monomers and fine-tune UGT activity. Glucuronide disposition may also be influenced by endoplasmic reticulum-localized β-glucuronidase, possibly involved in hydrolysis of hormone and drug glucuronides in target cells. The present commentary reviews recent advances and addresses open questions. Resolution of these questions may help to understand many problems of glucuronide synthesis and disposition in vivo, for example, under-prediction of the in vivo clearance of drugs mostly eliminated by glucuronidation by in vitro enzyme kinetic parameters of UGTs.
Characterization of rabbit UDP-glucuronosyltransferase UGT1A7: Tertiary amine glucuronidation is catalyzed by UGT1A7 and UGT1A4
1997, Archives of Biochemistry and Biophysics
A rabbit liver UDP-glucuronosyltransferase cDNA that is related to human and rat UGT1A7 has been identified. The predicted amino acid sequence of the UGT1A7l displays 80% similarity to that encoded by human HP4 (UGT1A9), but 81% to that predicted for human UGT1A7 and 77% to the rat UGT1A7 (UGTA2). The exons encoding human UGT1A7 and rat UGTA2 are the seventh of the series of cassette exons that flank the 3′ common exon series of theUGT1Alocus. Southern blot analysis demonstrates that the exon sequence encoding UGT1A7l is part of a larger cluster of highly related genes. The UGT1A7l RNA is expressed in both neonatal and adult liver, and unlike rat UGT1A2 which is inducible with Ah receptor ligands such as polycyclic aromatic hydrocarbons, rabbit UGT1A7l is not regulated when animals are exposed to these inducers. Following expression of UGT1A7l in COS-1 cells, glucuronidation activity was identified for small phenolic molecules like 4-nitrophenol, bulky phenols as represented by 4-hydroxybiphenol and octylgallate, as well as 4-hydroxyestrone. In addition, UGT1A7l possesses catalytic activity toward tertiary amines like the tricyclic antidepressant imipramine. The pattern of UGT1A7l glucuronidation is similar to that observed for human UGT1A9, except tertiary amines are not subject to glucuronidation by human UGT1A9. Glucuronidation of tertiary amines is catalyzed principally by human UGT1A4 as well as rabbit UGT1A4. Although rabbit UGT1A7l catalyzes the formation of quarternary ammonium glucuronides, theV_maxis considerably less than that observed for rabbit UGT1A4. Overall, the characterization of rabbit UGT1A7l suggests that this protein represents the ortholog of the human UGT1A7, which to date has not been identified.
The influence of age and some inducers on UDP-glucuronyltransferase activity
1997, Experimental Gerontology
UDP-glucuronyltransferase (UDP-GT) activity was examined in male Wistar rats aged 0.5, 1, 2, 4, 8, 12, 20, and 28 months. The rats were treated with phenobarbital (75 mg/kg, 72 and 48 h before death), β-naphthoflavone or dexamethasone (40 mg/kg and 20 mg/kg, respectively, for three days before death). Prior to decapitation the rats were fasted for 12 h. Hepatic microsomes were prepared according to the method of Dallner. UDP-GT activity was determined by the method of Burchell and Weatherill. p-Nitrophenol was used as an aglucone. UDP-GT activity decreased rapidly in the control rats aged from two weeks to four months. In the older control rats the activity tended to increase. Two-week-old rats treated with phenobarbital showed a slightly increased UDP-GT activity. In the older animals (up to one year) UDP-GT activity increased to 150% of the control value and stayed at this level in the remaining age groups. β-Naphthoflavone was a more potent inducer of UDP-GT than phenobarbital. The activity of β-naphthoflavone-induced UDP-GT was low in the youngest rats. It was about 180% in two-month-old rats and reached 260% of the control value in eight-month-old rats. Although the activity decreased in the older rats, it still exceeded 200%. Dexamethasone did not affect UDP-GT activity. Only in two-week-old and two-month-old rats did we observe a slight increase in the activity of UDP-GT.
Application of photoaffinity labeling with [11,12-<sup>3</sup>H]all-trans-retinoic acid to characterization of rat liver microsomal UDP-glucuronosyltransferase(s) with activity toward retinoic acid
1997, Biochemical and Biophysical Research Communications
[³H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [³H]all-trans-retinoic acid and [³²P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 μM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [³H]all-trans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes.
Purification of a phenobarbital-inducible UDP-glucuronosyltransferase isoform from dog liver which catalyzes morphine and testosterone glucuronidation
1996, Archives of Biochemistry and Biophysics
A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle dog treated with phenobarbital. Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The microsomes were solubilized with Emulgen 911 and applied on a column of hemisuccinate derivative of Sepharose 4B column which has been developed in our laboratory. An isoform of UGT in the eluate was purified further by chromatofocusing and UDP-hexanolamine-affinity chromatography. A purified enzyme, UGT_DOG-PB, was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but not toward chloramphenicol and the 6-hydroxyl group of morphine. The substrate specificity of UGT_DOG-PBis similar to that of stably expressed UGT2B1 which is considered a phenobarbital-inducible morphine UGT in the rat except that UGT_DOG-PBis capable of glucuronidating 4-nitrophenol but not chloramphenicol. The NH₂-terminus until the 30th residue of UGT_DOG-PBis highly homologous to UGT2B subfamily, and the NH₂-terminal 15 residues of UGT_DOG-PBare completely identical to those of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily.

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¹: Present address: Developmental Pharmacology Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20205.

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Purification and properties of rabbit liver estrone and p-nitrophenol UDP-glucuronyltransferases

Abstract

Biochem. Pharmacol

J. Biol. Chem

FEBS Lett

FEBS Lett

J. Biol. Chem

Arch. Biochem. Biophys

Anal. Biochem

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

J. Chromatogr

J. Biol. Chem

J. Biol. Chem