Evaluation of variation of glucuronidation activity and substrate specificity caused by difference in the proportion of expression level of UGT isozimes in living cells

本研究的目的是阐明体外和体内UGT活性差异的原因。然后，我们开发了一种测定活细胞中UGT活性的方法(接近体内模型)。方法：LLC-PK1细胞与底物在冰上孵育2小时。孵化后，细胞在37摄氏度的水浴中孵化，以便酶和转运蛋白工作。建立了稳定表达UGT1A9的HeLa细胞系，并将UGT2B4导入其中。表达UGT2B4的HeLa细胞的UGT1A9活性降低。这一结果表明UGT之间存在蛋白质-蛋白质相互作用。

The aim of this study was to clarify the factor of gap between UGT activity evaluated by in vitro and in vivo assay. Then we have developed an assay method to determine UGT activity in living cells (as near in vivo model). The method is as follows: LLC-PK1 cells were incubated with substrate for 2 hours on ice. After incubating, the cells were incubated on water bath at 37 degree Celsius for enzymes and transporters working. And then metabolites were extracellularly excreted and quantified.Furthermore, we established stable HeLa cell line expressing UGT1A9 and introduced UGT2B4 into it. UGT1A9 activity of HeLa cells expressing UGT2B4 reduced. This result indicated the protein-protein interaction between the UGTs.