Patten C J, Ishizaki H, Aoyama T, Lee M, Ning S M, Huang W, Gonzalez F J, Yang C S
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855.
Arch Biochem Biophys. 1992 Nov 15;299(1):163-71. doi: 10.1016/0003-9861(92)90258-x.
A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.
利用痘苗病毒表达系统，在哺乳动物细胞系中表达了编码人细胞色素P450 2E1的全长cDNA。免疫印迹分析表明，表达的蛋白与抗大鼠2E1的多克隆抗体发生反应，并与人肝微粒体中的P450 2E1共迁移。在同时含有细胞色素b5和NADPH：P450氧化还原酶的人细胞系Hep G2细胞中表达的P450 2E1，能够代谢几种已知的P450 2E1底物：N-亚硝基二甲胺（NDMA）、N-亚硝基甲基苄胺（NMBzA）、对硝基苯酚、苯酚和对乙酰氨基酚。NDMA去甲基化的表观Km和Vmax值分别为22μM和173 pmol/分钟/毫克微粒体蛋白。在不含b5的TK-143细胞中表达的P450 2E1，其Km和Vmax值分别为31μM和34 pmol/分钟/毫克微粒体蛋白。将纯化的大鼠肝b5掺入TK-143微粒体中，使Vmax增加了2.2倍，并使Km降至22μM。向Hep G2微粒体中添加b5导致Vmax增加了1.6倍，但对Km没有影响。在Hep G2细胞中表达的P450 2E1被证明能够代谢NMBzA，其Km为47μM，Vmax为213 pmol/分钟/毫克微粒体蛋白。添加b5可将Km降至27μM，但对Vmax没有影响。这些结果确凿地证明，P450 2E1是肝微粒体中观察到的低Km形式的NDMA去甲基酶和NMBzA去苄基酶的原因，并且这些活性受细胞色素b5的影响。
