Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation

Ishii, Yuji; Miyoshi, Akio; Watanabe, Rikiya; Tsuruda, Kazuoki; Tsuda, Minoru; Yamaguchi-Nagamatsu, Yuki; Yoshisue, Kunihiro; Tanaka, Mitsuko; Maji, Daisuke; Ohgiya, Satoru; Oguri, Kazuta

doi:10.1124/mol.60.5.1040

Cited by 67 publications

(52 citation statements)

References 37 publications

(43 reference statements)

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“…The interaction between the two monomers in dimeric UGT1A9 appears to be tight and stable, and they are not easily dissociated from each other under the conditions used for extraction from the membrane and affinity purification. In light of the present findings and previous reports (19,22,23), it may be suggested that dimer formation is indeed a property of many UGTs, and might be essential for UGTs activity. Nevertheless, detergent inhibition is not mediated by monomerization, at least as far as the entacapone glucuronidation activity of UGT1A9 is concerned.…”

Section: Discussionsupporting

confidence: 53%

“…It was recently suggested that heterodimers including the guinea pig enzymes UGT2B21 and UGT2B22 might exhibit activity that is either weaker or even undetectable when either of the two participating UGTs is expressed alone (22). On the other hand, it was reported that UGT1A1 forms homodimers and not heterodimers and that dimers that contain one native and one mutant monomer are partly or completely inactive (23).…”

Section: Udp-glucuronosyltransferases (Ugts)mentioning

confidence: 99%

“…Purification of an active UGT1A9 allows us to ask whether the UGTs are dimeric enzymes, as suggested previously (19,22,23). However, determination of the molecular weight of a purified membrane enzyme, e.g.…”

Section: Table III Effect Of Triton X-100 (02% W/v) On the Glucuronmentioning

confidence: 99%

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Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Kurkela¹,

Garcı́a-Horsman²,

Luukkanen³

et al. 2003

Journal of Biological Chemistry

138

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Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of ␣-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with Histagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.

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Section: Discussionsupporting

confidence: 53%

Section: Udp-glucuronosyltransferases (Ugts)mentioning

confidence: 99%

See 1 more Smart Citation

Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Kurkela¹,

Garcı́a-Horsman²,

Luukkanen³

et al. 2003

Journal of Biological Chemistry

138

View full text Add to dashboard Cite

show abstract

“…Ishii and colleagues have found in fact that the simultaneous expression of the isoenzymes UGT2B21 and UGT2B22 in guinea pigs produces extensive M6G formation (Ishii et al, 2001). The hypothesis that heroin induced the synthesis of M6G by modulating the formation of heterooligomers of UGT is speculative but deserves to be further investigated.…”

Section: Discussionmentioning

confidence: 99%

Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat

Antonilli¹,

Suriano²,

Paolone³

et al. 2003

J Pharmacol Exp Ther

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After absorption, heroin is transformed into mono-acetyl-morphine and then into morphine. Morphine, in turn, is metabolized to morphine-3-glucuronide (M3G), an inactive compound, and morphine-6-glucuronide (M6G), a potent opioid agonist. Thus, changes in the rate of formation of M6G may alter the pharmacological consequences of a treatment with heroin or morphine. In this study, we investigate the effect of repeated exposures (10 daily i.p. injections) to heroin, morphine, cadmium (which has been previously shown to inhibit M3G formation in vitro), or heroin ϩ cadmium on morphine glucuronidation both in vivo and ex vivo (i.e., microsomal preparation obtained from rats treated in vivo). Repeated heroin (2.5, 5.0, and 10 mg/kg) increased plasma levels of M6G (which was undetectable in all other groups) and reduced those of M3G. Also, the microsomal preparations obtained from the liver of repeated heroin rats, when incubated with morphine, yielded significant amounts of M6G (which was undetectable in all other groups) and decreased levels of M3G relative to the control groups. These effects were reversible upon discontinuation of heroin administration. In contrast, repeated morphine (10, 20, and 40 mg/kg) only slightly reduced M3G formation at the dose of 40 mg/kg. Repeated cadmium (5, 15, and 45 g/kg) reduced the rate of M3G formation without inducing M6G synthesis. The effects of the repeated coadministration of heroin (10 mg/kg) and cadmium (15 g/kg) were virtually identical to those of repeated heroin alone. In summary, repeated exposure of rats to heroin can shift morphine glucuronidation toward the formation of the active metabolite M6G.

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“…Homooligomer formation of rat UGT2B1 (Meech and Mackenzie, 1997) and hetero-oligomer formation of rat UGT2B1 and UGT1A family (Ikushiro et al, 1997) have been reported. Recently, Ishii et al (2001) reported that hetero-oligomer formation of guinea pig UGT2B21 and UGT2B22 leads to altered substrate specificity. In recombinant UGTs used in most studies, including the present study, only homo-oligomers would be formed.…”

Section: Correlation Coefficients Between Nicotine N-glucuronidationmentioning

confidence: 99%

Characterization of Nicotine and CotinineN-Glucuronidations in Human Liver Microsomes

Nakajima¹,

Tanaka²,

Kwon³

et al. 2002

Drug Metab Dispos

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ABSTRACT:The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K m and V max values were 33.1 ؎ 28.1 M and 60.0 ؎ 21.0 pmol/ min/mg and 284.7 ؎ 122.0 M and 124.0 ؎ 44.0 pmol/min/mg for the high-and low-affinity components, respectively, in human liver microsomes (n ‫؍‬ 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K m ‫؍‬ 1.9 ؎ 0.3 mM, V max ‫؍‬ 655.6 ؎ 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K i ‫؍‬ 3.9 and 3.3 M), imipramine as a substrate for UGT1A4 (K i ‫؍‬ 6.1 and 2.7 M), and propofol as a substrate for UGT1A9 (K i ‫؍‬ 6.0 and 12.0 M). The nicotine N-glucuronidation (50 M nicotine) in 14 human liver microsomes was significantly (r ‫؍‬ 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r ‫؍‬ 0.425) or cotinine N-glucuronidation (r ‫؍‬ 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.

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Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation

Cited by 67 publications

References 37 publications

Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat

Characterization of Nicotine and CotinineN-Glucuronidations in Human Liver Microsomes

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