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A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue

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A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue. / Milne, Alison M.; Burchell, Brian; Coughtrie, Michael W. H.

In: Drug Metabolism and Disposition, Vol. 39, No. 12, 2011, p. 2258-2263.

Research output: Contribution to journalArticle

Harvard

Milne, AM, Burchell, B & Coughtrie, MWH 2011, 'A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue' Drug Metabolism and Disposition, vol 39, no. 12, pp. 2258-2263., 10.1124/dmd.111.041699

APA

Milne, A. M., Burchell, B., & Coughtrie, M. W. H. (2011). A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue. Drug Metabolism and Disposition, 39(12), 2258-2263. 10.1124/dmd.111.041699

Vancouver

Milne AM, Burchell B, Coughtrie MWH. A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue. Drug Metabolism and Disposition. 2011;39(12):2258-2263. Available from: 10.1124/dmd.111.041699

Author

Milne, Alison M.; Burchell, Brian; Coughtrie, Michael W. H. / A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue.

In: Drug Metabolism and Disposition, Vol. 39, No. 12, 2011, p. 2258-2263.

Research output: Contribution to journalArticle

Bibtex - Download

@article{b2fdd5493f0b43a19fe372c476d0d7de,
title = "A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue",
keywords = "Absolute quantification, Gilberts syndrome, Escherichia coli, Interindividual variability, Human sulfotransferases, Liver microsomes, Glucuronidation, Expression, Gene, Drug",
author = "Milne, {Alison M.} and Brian Burchell and Coughtrie, {Michael W. H.}",
year = "2011",
doi = "10.1124/dmd.111.041699",
volume = "39",
number = "12",
pages = "2258--2263",
journal = "Drug Metabolism and Disposition",
issn = "0090-9556",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue

A1 - Milne,Alison M.

A1 - Burchell,Brian

A1 - Coughtrie,Michael W. H.

AU - Milne,Alison M.

AU - Burchell,Brian

AU - Coughtrie,Michael W. H.

PY - 2011

Y1 - 2011

N2 - <p>Glucuronidation is a major pathway of drug and xenobiotic metabolism that is catalyzed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards because of the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in Escherichia coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. After addition of ribonuclease S-protein to membrane extracts, a functional ribonuclease is reconstituted that provides a direct and absolute quantification of the amount of UGT fusion protein; this is subsequently used to generate standard curves for immunoquantification by immunoblotting. To illustrate the value of the method, we have quantified the expression of UGT1A1 and UGT1A6 in human liver and kidney microsomes using new isoform-specific antibodies developed against peptides from these proteins. Expression levels of both proteins in liver were highly variable (28- and 20-fold, respectively) and correlated strongly with UGT enzyme activity toward the probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining the absolute, as opposed to relative, quantities of UGT proteins present in human tissues.</p>

AB - <p>Glucuronidation is a major pathway of drug and xenobiotic metabolism that is catalyzed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards because of the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in Escherichia coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. After addition of ribonuclease S-protein to membrane extracts, a functional ribonuclease is reconstituted that provides a direct and absolute quantification of the amount of UGT fusion protein; this is subsequently used to generate standard curves for immunoquantification by immunoblotting. To illustrate the value of the method, we have quantified the expression of UGT1A1 and UGT1A6 in human liver and kidney microsomes using new isoform-specific antibodies developed against peptides from these proteins. Expression levels of both proteins in liver were highly variable (28- and 20-fold, respectively) and correlated strongly with UGT enzyme activity toward the probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining the absolute, as opposed to relative, quantities of UGT proteins present in human tissues.</p>

KW - Absolute quantification

KW - Gilberts syndrome

KW - Escherichia coli

KW - Interindividual variability

KW - Human sulfotransferases

KW - Liver microsomes

KW - Glucuronidation

KW - Expression

KW - Gene

KW - Drug

U2 - 10.1124/dmd.111.041699

DO - 10.1124/dmd.111.041699

M1 - Article

JO - Drug Metabolism and Disposition

JF - Drug Metabolism and Disposition

SN - 0090-9556

IS - 12

VL - 39

SP - 2258

EP - 2263

ER -

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