Yamazaki H, Nakano M, Gillam E M, Bell L C, Guengerich F P, Shimada T
Osaka Prefectural Institute of Public Health, Japan.
Biochem Pharmacol. 1996 Jul 26;52(2):301-9. doi: 10.1016/0006-2952(96)00208-0.
NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.
在含有细胞色素b5（b5）、NADH - b5还原酶和细菌重组P450 2E1的体系中，于含合成磷脂混合物和胆酸盐的100 mM磷酸钾缓冲液（pH 7.4）中可重建依赖NADH的7 - 乙氧基香豆素O - 脱乙基活性。用NADPH - P450还原酶替代NADH - b5还原酶，7 - 乙氧基香豆素O - 脱乙基活性增加4倍，当使用NADPH作为电子供体时，活性可进一步受到刺激（约1.5倍）。在存在NADPH - P450还原酶的情况下，从NADH和NADPH支持的体系中去除b5导致7 - 乙氧基香豆素O - 脱乙基活性损失90%，但在不存在NADPH - P450还原酶时导致活性完全丧失。当从NADPH支持的P450 2E1重建体系中省略b5时，7 - 乙氧基香豆素O - 脱乙基的Km值增加而Vmax值降低。在氯唑沙宗6 - 羟基化、苯胺对羟基化和N - 亚硝基二甲胺N -去甲基化反应中也观察到P450 2E1体系对b5的需求。在人肝微粒体中，发现依赖NADH的7 - 乙氧基香豆素O - 脱乙基、氯唑沙宗6 - 羟基化、苯胺对羟基化和N - 亚硝基二甲胺N - 去甲基化活性分别约为NADPH支持体系催化活性的55%、41%、33%和50%。抗大鼠NADPH - P450还原酶免疫球蛋白G在NADPH支持的反应中比在NADH支持的反应中更强烈地抑制人肝微粒体催化的7 - 乙氧基香豆素O - 脱乙基活性，而抗人b5免疫球蛋白G在NADH和NADPH支持的体系中对微粒体活性的抑制程度相似。这些结果表明，b5是P450 2E1催化几种所用底物氧化的必需成分，约10%的活性在不存在b5时通过NADPH - P450还原酶对P450 2E1的还原发生，并且NADH支持的体系部分地参与人肝微粒体中P450 2E1催化的一些反应。
