Fujiwara Ryoichi, Nakajima Miki, Yamanaka Hiroyuki, Katoh Miki, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-1192, Japan.
Drug Metab Dispos. 2007 Oct;35(10):1781-7. doi: 10.1124/dmd.107.016402. Epub 2007 Jul 9.
Protein-protein interactions between human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A4, and UGT1A6 were investigated using double expression systems in HEK293 cells (UGT1A1/UGT1A4, UGT1A1/UGT1A6, and UGT1A4/UGT1A6). The substrates specific for UGT1A1 (estradiol and bilirubin), UGT1A4 (imipramine and trifluoperazine), and UGT1A6 (serotonin and diclofenac) were used to determine the effects of the coexpression of the other UGT1A isoforms on the enzymatic activity. The coexpression of UGT1A4 and UGT1A6 decreased the S(50) and V(max) values of UGT1A1-catalyzed estradiol 3-O-glucuronide formation and increased the V(max) value of UGT1A1-catalyzed bilirubin O-glucuronide formation. The coexpression of UGT1A1 decreased the V(max) value of UGT1A4-catalyzed imipramine N-glucuronide formation but had no effect on UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of UGT1A6 had no effect on UGT1A4-catalyzed imipramine N-glucuronide formation but increased the K(m) and V(max) of UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of both UGT1A1 and UGT1A4 increased the V(max) values of UGT1A6-catalyzed serotonin and diclofenac O-glucuronide formation. Thus, the effects of the coexpression of other UGT1A isoforms on the kinetics of specific activities were different depending on the UGT1A isoforms and substrates. Native polyacrylamide gel electrophoresis analysis of the double expression systems showed multiple bands at approximately 110 kDa, indicating the existence of heterodimers as well as homodimers of UGTs. In conclusion, we found that human UGT1A1, UGT1A4, and UGT1A6 interact with each other, possibly by heterodimerization, and that their effects on the enzymatic activities are complex depending on the isoforms and substrates.
利用HEK293细胞中的双表达系统（UGT1A1/UGT1A4、UGT1A1/UGT1A6和UGT1A4/UGT1A6）研究了人尿苷二磷酸葡萄糖醛酸基转移酶（UGT）1A1、UGT1A4和UGT1A6之间的蛋白质-蛋白质相互作用。使用UGT1A1的特异性底物（雌二醇和胆红素）、UGT1A4的特异性底物（丙咪嗪和三氟拉嗪）以及UGT1A6的特异性底物（5-羟色胺和双氯芬酸）来确定其他UGT1A同工型的共表达对酶活性的影响。UGT1A4和UGT1A6的共表达降低了UGT1A1催化雌二醇3-O-葡萄糖醛酸苷形成的S(50)和V(max)值，并增加了UGT1A1催化胆红素O-葡萄糖醛酸苷形成的V(max)值。UGT1A1的共表达降低了UGT1A4催化丙咪嗪N-葡萄糖醛酸苷形成的V(max)值，但对UGT1A4催化三氟拉嗪N-葡萄糖醛酸苷形成没有影响。UGT1A6的共表达对UGT1A4催化丙咪嗪N-葡萄糖醛酸苷形成没有影响，但增加了UGT1A4催化三氟拉嗪N-葡萄糖醛酸苷形成的K(m)和V(max)。UGT1A1和UGT1A4的共表达均增加了UGT1A6催化5-羟色胺和双氯芬酸O-葡萄糖醛酸苷形成的V(max)值。因此，其他UGT1A同工型共表达对比活性动力学的影响因UGT1A同工型和底物而异。对双表达系统进行的天然聚丙烯酰胺凝胶电泳分析显示，在约110 kDa处有多个条带，表明存在UGT的异二聚体以及同二聚体。总之，我们发现人UGT1A1、UGT1A4和UGT1A6相互作用，可能是通过异二聚化，并且它们对酶活性的影响因同工型和底物而异，情况较为复杂。
