Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
J Pharm Sci. 2010 Jan;99(1):442-54. doi: 10.1002/jps.21830.
Glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT) enzymes is an important pathway in the metabolism of drugs as well as environmental chemicals. In this study, protein-protein interactions between human UGT2B7 and UGT1As and their effects on the enzymatic activities were investigated using double expression systems in HEK293 cells (UGT2B7/UGT1A1, UGT2B7/UGT1A4, UGT2B7/UGT1A6, and UGT2B7/UGT1A9). Native-PAGE analysis clearly revealed that UGT2B7 forms homo-oligomers. Furthermore, hetero-oligomers of UGT2B7 with UGT1As were observed by native-PAGE analysis. Immunoprecipitation assay revealed associations of UGT2B7 with UGT1A1, UGT1A4, UGT1A6, and UGT1A9. The thermal stability of UGT2B7 was significantly increased by the coexpressed UGT1A1, UGT1A4, UGT1A6, and UGT1A9, indicating an interaction between UGT2B7 and the UGT1As. To examine the effects of the protein-protein interactions on the enzymatic activities, kinetic analyses were performed. Coexpression of the UGT1As significantly decreased K(m) and increased V(max) of zidovudine O-glucuronidation by UGT2B7. Coexpression of UGT2B7 also affected the kinetics of estradiol 3-O-glucuronidation by UGT1A1, imipramine N-glucuronidation by UGT1A4, serotonin O-glucuronidation by UGT1A6, and propofol O-glucuronidation by UGT1A9. In conclusion, it was clearly demonstrated that human UGT2B7 interacts with UGT1A enzymes, affecting their kinetics. That such interactions might occur in human liver microsomes underscores the complexities in glucuronidations in human liver.
尿苷二磷酸葡萄糖醛酸基转移酶（UGT）催化的葡萄糖醛酸化是药物和环境化学物质代谢的重要途径。在这项研究中，使用 HEK293 细胞中的双表达系统（UGT2B7/UGT1A1、UGT2B7/UGT1A4、UGT2B7/UGT1A6 和 UGT2B7/UGT1A9）研究了人 UGT2B7 与 UGT1As 之间的蛋白质-蛋白质相互作用及其对酶活性的影响。天然 PAGE 分析清楚地表明 UGT2B7 形成同型寡聚体。此外，通过天然 PAGE 分析观察到 UGT2B7 与 UGT1As 的异型寡聚体。免疫沉淀试验表明 UGT2B7 与 UGT1A1、UGT1A4、UGT1A6 和 UGT1A9 相关联。UGT1A1、UGT1A4、UGT1A6 和 UGT1A9 的共表达显著增加了 UGT2B7 的热稳定性，表明 UGT2B7 与 UGT1As 之间存在相互作用。为了研究蛋白质-蛋白质相互作用对酶活性的影响，进行了动力学分析。UGT1As 的共表达显著降低了 UGT2B7 对扎西他滨 O-葡萄糖醛酸化的 K(m)并增加了 V(max)。UGT2B7 的共表达也影响了 UGT1A1 对雌二醇 3-O-葡萄糖醛酸化、UGT1A4 对丙咪嗪 N-葡萄糖醛酸化、UGT1A6 对 5-羟色胺 O-葡萄糖醛酸化和 UGT1A9 对丙泊酚 O-葡萄糖醛酸化的动力学。总之，本研究清楚地表明人 UGT2B7 与 UGT1A 酶相互作用，影响其动力学。这些相互作用可能发生在人肝微粒体中，强调了人肝中葡萄糖醛酸化的复杂性。
