title: A novel method for the immunoquantification of UDP-glucuronosyltransferases in human tissue.
abstract: 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.
authors: Division of Medical Sciences, University of Dundee, Ninewells Hospital & Medical School, Dundee DD1 9SY, Scotland, UK.
authors: Alison M
authors: AM
authors: Milne
authors: Brian
authors: B
authors: Burchell
authors: Michael W H
authors: MW
authors: Coughtrie
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D014453:Glucuronosyltransferase