| 8161205 |
Ouzzine M, Pillot T, Fournel-Gigleux S, Magdalou J, Burchell B, Siest G: Expression and role of the human liver UDP-glucuronosyltransferase UGT1*6 analyzed by specific antibodies raised against a hybrid protein produced in Escherichia coli. Arch Biochem Biophys. 1994 Apr;310(1):196-204.
Characterization of human UDP-glucuronyltransferases (UGTs) has been limited by the unavailability of probes selective for each of several highly related isoforms. To better understand the role of this superfamily in the metabolism of drugs and xenobiotics, we describe a molecular/immunological strategy for discriminating the implication of each human isoenzyme in this process. Specific polyclonal antibodies were generated against the divergent amino-terminal domain of the UGT isoform UGT1*6 which is involved in the detoxification of nucleophilic compounds related to phenols and naphthols in human liver. The novel approach consists of the expression of a N-terminal UGT polypeptide fused to Staphylococcus aureus protein A in Escherichia coli and a single step purification of the fusion protein by immunoaffinity chromatography. Immunoblot and immunoinhibition analysis showed that the antibodies raised against the fusion protein selectively recognized both the denaturated and the native forms of UGT1*6, when expressed in V79 cell lines, but not three other recombinant UGT isoenzymes. In human liver microsomes, specific immunoinhibition analysis demonstrated that glucuronidation by UGT1*6 represented 20 to 50% of the total 1-naphthol UGT activity with a good correlation with the amount of protein selectively quantified on immunoblot. The specific expression of UGT1*6 was found to be significantly reduced in tumoral tissues but enhanced in cholestatic livers, when compared with healthy hepatic tissues. Interestingly, in human kidney microsomes, antibodies revealed a high level of UGT1*6 expression on immunoblot and inhibited 1-naphthol glucuronidation up to 55%, indicating that this isoform is also expressed in kidney and extensively contributes to phenol glucuronidation in this tissue. |
115(1,2,2,5) |