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

活细胞中UGT同工酶表达水平比例差异引起的葡萄糖醛酸化活性和底物特异性变化的评价

• 批准号: 24790139
• 负责人: TAKEKUMA Yoh
• 金额: $ 2.91万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2012
• 资助国家: 日本
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-24790139/
• 关键词: UGT; 生細胞; グルクロン酸抱合; 薬物代謝; グルクロン酸転移酵素

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.

本研究的目的是阐明UGT活性之间的差距的因素，在体外和体内测定评价。然后，我们开发了一种测定活细胞中UGT活性的测定方法（接近体内模型）。方法如下：LLC-PK 1细胞与底物在冰上孵育2小时。孵育后，将细胞在37摄氏度的水浴上孵育，以使酶和转运蛋白发挥作用。建立了稳定表达UGT 1A 9的HeLa细胞系，并将UGT 2B 4导入其中，表达UGT 2B 4的HeLa细胞的UGT 1A 9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。

项目成果

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