Modulation of UDP-glucuronosyltransferase function by cytochrome P450: Evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4
Modulation of UDP-glucuronosyltransferase function by cytochrome P450: Evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4
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DOI：
10.1124/mol.104.007641
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Yamada, H
中科院分区：
文献类型：
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作者：
Takeda, S;Ishii, Y;Yamada, H
研究了细胞色素P450(P450)对UDP-葡萄糖醛酸基转移酶2B7(UGT2B7)催化吗啡葡萄糖醛酸化反应的调节作用。通过在COS-1细胞中同时表达UGT2B7和CYP3A4、-1A2或-2C9，研究了P450同工酶对UGT2B7催化的吗啡3-羟基葡萄糖醛酸化反应动力学参数的影响。虽然与UGT2B7共表达对V-max的影响不大，但与UGT2B7单表达系统相比，K-m增加了约9.8倍。另两种P450同工酶(CYP1A2和CYP2C9)对K-m和V-max有一定的影响。从溶解的人肝微粒体中免疫共沉淀UGT导致了CYP3A4与UGT2B7的共沉淀。用谷胱甘肽S-转移酶-细胞色素P3A4融合蛋白的重叠实验进一步证实了细胞色素P3A4和UGT2B7之间的蛋白质相互作用。在未处理的表达UGT2B7的COS微粒体中加入CYP3A4对吗啡的葡萄糖糖醛酸化反应无影响或影响很小。反之，经洗涤剂处理后的COS-1细胞表达UGT2B7的微粒体内吗啡-3-葡萄糖醛酸苷的生成被CYP3A4抑制，而6-葡萄糖醛酸苷的生成则被促进。这些结果有力地表明：1)UGT2B7对吗啡的葡萄糖醛酸化活性是通过与微粒体膜上的CYP3A4相互作用来特异调节的；2)CYP3A4改变了UGT2B7的区域选择性，从而提高了吗啡的激活/解毒比。这项研究首次证明P450不仅参与药物的氧化，而且还调节UGT的功能。
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.