Modulation of UDP-glucuronosyltransferase 2B7 (UGT2B7)-catalyzed morphine glucuronidation by cytochrome P450 (P450) was studied. The effects of P450 isozymes on the kinetic parameters of UGT2B7-catalyzed glucuronidation of the morphine 3-hydroxyl group were examined by simultaneous expression of UGT2B7 and either CYP3A4, -1A2, or -2C9 in COS-1 cells. Although coexpression of CYP3A4 with UGT2B7 had little effect on V max , the K m was increased by about 9.8-fold compared with the UGT2B7 single expression system. The other P450 isozymes (CYP1A2 and CYP2C9) had some effects on K m and V max values. Immunoprecipitation of UGT from solubilized human liver microsomes resulted in coprecipitation of CYP3A4 with UGT2B7. The protein-protein interaction between CYP3A4 and UGT2B7 was further confirmed by overlay assay using glutathione S-transferase-CYP3A4 fusion protein.Addition of CYP3A4 to untreated COS microsomes expressing UGT2B7 had no or minor effects on morphine glucuronidation. In contrast, the formation of morphine-3-glucuronide by detergent-treated microsomes from COS-1 cells expressing UGT2B7 was reduced by CYP3A4, whereas the formation of the 6-glucuronide was enhanced. These results strongly suggest that 1) the glucuronidation activity of UGT2B7 toward morphine is specifically modulated by interaction with CYP3A4 in microsomal membranes and that 2) CYP3A4 alters UGT2B7 regioselectivity so that the ratio of morphine activation/detoxication is increased. This study provides the first evidence that P450 is not only involved in oxidation of drugs but also modulates the function of UGTs.

We have reported that the protein-protein interaction between UDP-glucuronosyltransferase (UGT) 2B7 and cytochrome P450 3A4 (CYP3A4) alters UGT2B7 function. However, the domain(s) involved in the interaction are largely unknown. To address this issue, we examined in more detail the CYP3A4-UGT2B7 association by means of immunoprecipitation, overlay assay, and cross-linking involving 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Purified CYP3A4 or glutathione transferase (GST)-tagged CYP3A4 was cross-linked to UGT2B7 in solubilized baculosomes. The formation of the cross-linked complex was detected by immunoblotting using both antibodies against CYP3A4 and UGTs. Although the GST-tagged CYP3A4 containing the region ranging from Tyr25 to Ala503 was cross-linked to UGT2B7, the same did not occur when another construct containing Met145 to His267 was used. This observation was consistent with the result of the overlay assay indicating that CYP3A4 lacking the N-terminal hydrophobic segment retains the ability to associate with UGT2B7, whereas the Met145-to-His267 region loses this capacity. Although the Met145-to-His267 peptide was recognized by one anti-CYP3A4 antibody that has the ability to coimmunoprecipitate UGT2B7, it was not recognized by another antibody incapable of coimmunoprecipitating UGT2B7. The epitope of the latter antibody was mapped to the Leu331-to-Lys342 region, which is located on the J-helix of CYP3A4. Taken together, the results obtained suggest that 1) CYP3A4 and UGT2B7 are a pair of enzymes in proximity to each other and 2) either the Leu331-to-Lys342 domain or the surrounding region plays a role in the interaction with UGT2B7, whereas the hydrophobic Met145-to-His267 region does not contribute to this interaction.Cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) are two major enzyme groups responsible for phase I and II reactions, respectively (Guengerich, 1989;Oguri et al., 1994;Gonzalez and Lee, 1996;Ritter, 2000). These enzymes are localized on the cytosolic (P450) and luminal sides (UGT) of the endoplasmic reticulum (ER) membrane. UGT plays an important role in detoxifying drugs, including potent carcinogenic metabolites formed by P450 (Tukey and Strassburg, 2000). To minimize toxicity, it would be reasonable to expect that the reactive metabolite produced by P450 is directly transferred to the other enzymes participating in its subsequent metabolism (e.g., the UGTs) via protein-protein interactions. Our recent studies have suggested that CYP3A4 and other P450 isoforms interact with UGT2B7 to modulate the activity of the UGT (Ishii et al., 2005; Takeda et al., 2005a,b). The interactions between P450 and UGT occur also in rats, and the UGT in a P450-UGT complex is catalytically active (Ishii et al., 2007). These series

The interaction between cytochrome P450s (CYP, P450) and UDP-glucuronosyltransferases (UGTs) was studied by co-immunoprecipitation. P450 isoform-selective antibody was used as a probe to co-precipitate UGTs with the P450s from solubilized rat liver microsomes. Antibodies toward CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 co-precipitated UGTs with corresponding P450s. However, calnexin, a type-I membrane protein, in the endoplasmic reticulum was not co-precipitated by anti-P450 antibodies. UGT activity toward 4-methylumbelliferone was detected in all co-precipitates, suggesting that UGT in the complex with P450s is functionally active. Repeated washing of co-immunoprecipitates revealed differences among P450 isoforms with regard to the affinity for UGT. Larger amounts of UGT1A1 and UGT1A6, compared with UGT2B1, were washed out from UGTs-CYP2C11/13 co-precipitates, whereas UGT-CYP3A2 and UGT-CYP2Bs complexes were resistant to thorough washing. Thus, CYP2C11/13 could associate with UGTs, but the affinity is assumed to be weaker than that of CYP2B/3As. These results suggest that there is isoform specificity in the interaction between P450s and UGTs.

Conversion of γ-hydroxybutyric acid (GHB) to a fluorescent derivative using 3-bromomethyl-6,7-dimethoxy-1-methyl-1,2-dihydroquinoxaline-2-one (Br-DMEQ), and its application to drug screening were studied. Br-DMEQ reacted with the carboxyl group of sodium GHB in the presence of a potassium salt and crown ether to produce a fluorescent derivative, which could be easily detected by thin-layer chromatography (TLC). An electrospray ionization mass spectrum of the fluorogenic product supported the expected structure. The Br-DMEQ-derivatized GHB gave an Rf value of 0.49 on TLC, which was easily distinguished from 19 other carboxylic acids; all of the latter had Rf values over 0.61. Various sodium carboxylates including sodium GHB reacted with Br-DMEQ in the presence of KCl, but the free forms of the carboxylic acids did not react under these conditions. When aqueous solution containing GHB was pretreated with sulfuric acid, GHB was converted to γ-butyrolactone, resulting in removal of its reactivity with Br-DMEQ. By such analysis following the derivatization, with and without the above pretreatment with sulfuric acid, GHB added to human urine could be specifically detected, although the limit of detection was about 100 µg/ml, which was 20 times higher than the endogenous blood GHB level. Therefore, the present method seems useful for screening GHB present in a solid state such as powder and tablets, and also in urine samples obtained in overdose cases.