7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.
7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.
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DOI：
10.1016/0006-2952(95)02178-7
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发表时间：
1996-02
影响因子：
5.8
通讯作者：
H. Yamazaki;K. Inoue;M. Mimura;Y. Oda;F. Guengerich;T. Shimada
中科院分区：
文献类型：
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作者：
H. Yamazaki;K. Inoue;M. Mimura;Y. Oda;F. Guengerich;T. Shimada
7-乙氧基香豆素O-脱乙基化已被广泛用作评价哺乳动物肝微粒体中细胞色素P450（P450）底物特异性的标记活性，广泛的研究表明，在大鼠和小鼠中，主要催化剂是P450 1A 1、1A 2和2B酶。与实验动物模型中的发现相反，据报道P450 2 E1是人体肝脏中参与7-乙氧基香豆素O-脱乙基化的主要酶。在这项研究中，我们进一步研究了个人形式的人P450参与7-乙氧基香豆素O-脱乙基化的作用，使用微粒体从不同的人肝样品和人淋巴母细胞表达人P450酶和纯化的P450酶分离的大肠杆菌膜表达修饰的人P450蛋白。动力学分析表明，在不同的人体样本中，至少有两种不同的酶参与7-乙氧基香豆素O-脱乙基反应。在肝微粒体中含有大量P450 2 E1的样品显示O-脱乙基化的双相曲线，具有相对高的转换数，而富含P450 1A 2的样品往往具有低Km值和低Vmax值。抗人P450 2 E1抗体显著抑制人肝微粒体催化的7-乙氧基香豆素O-脱乙基反应（P < 0.05），尤其是在高底物浓度（200 μM）下。然而，我们还发现抗P450 1A 2抗体仅在低底物浓度（10 μM）下抑制O-脱乙基活性。重组人P450 1A 2对7-乙氧基香豆素O-脱乙基反应的Km值较低，而P450 2 E1的Km值较高。P450酶检查，P450 1A 1给出了最高的O-脱乙基化活性与低Km值，虽然这种酶被报道在人类肝外表达。其他人P450酶，包括P450 2A 6，2C 10，2D 6，3A 4和3A 5，没有显示出显着的O-脱乙基活性，除了P450 2B 6，人肝脏中的一种次要P450组分，被发现具有与P450 1A 2相似的Vmax值和与P450 2 E1相似的Km值。这些结果表明，P450 1A 2是人肝微粒体中7-乙氧基香豆素O-脱乙基化的低Kmenzyme，尽管其Vmax值低于P450 2 E1。
7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from membranes of Escherichia coli expressing modified human P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Kmvalues with low Vmaxvalues. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 μM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 μM). Recombinant human P450 1A2 was found to have a low Kmvalue for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Kmvalue. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Kmvalue, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmaxvalue similar to that of P450 1A2 and a Kmvalue similar to that of P450 2E1. These results suggest that P450 1A2 is a low Kmenzyme for 7-ethoxycoumarin O-deethylation in human liver microsomes, although it has a lower Vmaxvalue than P450 2E1.