UDP-glucuronosyltransferase (UGT) contributes to the inactivation of a number of exogenous and endogenous compounds by conjugating them with glucuronic acid.1,2) The UGT isoforms are separated into two families (UGT1 and UGT2), based on the sequence identities.3) These UGTs show an overlapping but distinct substrate specificity, and are expressed in an organ-specific manner [see review of Tukey and Strassburg 4) ]. Increasing evidence suggests that there is a change in the catalytic function of UGT produced by oligomer formation of UGT isoforms.5-7) A recent study from this laboratory also revealed that glucuronidation of morphine at the 6-position by UGT2B21 expressed in COS cells is greatly enhanced by co-expression of a different isoform, UGT2B22. 8) Thus, UGT function appears to be changed dynamically through protein-protein interactions.UGT is anchored to the endoplasmic reticulum (ER) membrane by the transmembrane domain near the C-terminal, and the main body of the enzyme, including the catalytic site, is believed to be present in the lumen of the ER.2,9,10) This mode of topology causes latency, a diagnostic feature of UGT, whereby UGT activity is activated by disruption of the microsomal membrane with detergent.11) On the other hand, Ikushiro et al. (1997) 12) have reported that formation of UGT hetero-oligomer facilitates the transport of UDP-glucuronic acid from the cytosol to the inside of the ER. This observation suggests that a putative UDP-glucuronic acid transporter, which is expressed in the membrane, is activated by UGT oligomer. If this is the case, UGT would be partially embedded in ER membrane so that it can interact with the transporter. In clear contrast to UGT, an entire molecule containing the functional domain of cytochrome P450 (P450, CYP), which is anchored by the N-terminal region to the ER, is present in cytosol. [13][14][15] It has, however, been reported that P450 is partially drawn into the lipid bilayer membrane to change its secondary structure and increase its catalytic activity.16) It remains unclear whether functional interactions between P450 and UGT exist. Our previous study provided direct evidence for possible interactions between CYP1A1 and other microsomal enzymes using affinity chromatography with a CYP1A1-conjugated column. 17) In that study, CYP1A1-UGTs and CYP1A1-microsomal epoxide hydrolase (mEH) interactions were suggested. Recently, we have demonstrated that mEH function is significantly enhanced by multiple forms of P450.18) Thus, microsomal drug metabolizing enzymes can interact with each other so that they rapidly convert exogenous compounds to highly polar metabolites which are easily excreted. However, to the best of our knowledge, it has never before been proven that P450 modifies UGT function via a protein-protein interaction. Although the membrane topology of these enzymes is quite different, several forms of UGT were effectively trapped on a CYP1A1-conjugated column.17) Thereby, it may be that there is functional co-operation between these enzymes.In thi...