Abstract
Glucuronidation is a major pathway of drug and xenobiotic metabolism, catalysed 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 due to 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 E. coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. Following 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 UGTs 1A1 and 1A6 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-fold and 20-fold, respectively), and correlated strongly with UGT enzyme activity towards probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining absolute, as opposed to relative, quantities of UGT proteins present in human tissues.
- Received July 11, 2011.
- Accepted August 31, 2011.
- The American Society for Pharmacology and Experimental Therapeutics