CATALYTIC PROPERTIES OF THE HUMAN CYTOCHROME-P450 2E1 PRODUCED BY CDNA EXPRESSION IN MAMMALIAN-CELLS
CATALYTIC PROPERTIES OF THE HUMAN CYTOCHROME-P450 2E1 PRODUCED BY CDNA EXPRESSION IN MAMMALIAN-CELLS
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DOI：
10.1016/0003-9861(92)90258-x
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发表时间：
1992-11-15
影响因子：
3.9
通讯作者：
YANG, CS
中科院分区：
文献类型：
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作者：
PATTEN, CJ;ISHIZAKI, H;YANG, CS
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c DNA。免疫印迹分析表明，表达蛋白能与大鼠2E1多克隆抗体反应，并能与人肝微粒体中的P450 2E1融合。在同时含有细胞色素b5和NADPH：P450氧化还原酶的人细胞株Hep G2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-亚硝基甲基苯甲胺(NMBzA)、对硝基苯酚、苯酚和对乙酰氨基酚。脱甲基化的KmandVmax分别为22pmolm和173pmoL/μ/mg微粒体蛋白。在不含b5的TK143细胞中表达的P4502e1的KmandVmax分别为31pmolm和34pmoL/μ/mg。将纯化的大鼠肝B5掺入TK−143型微体，使Vmax增加2.2倍，Km降低至22μm，加入B5可使Vmax增加1.6倍，但对Km无影响。在HEP G2细胞中表达的P4502E1代谢NMBzA，其AKm为47min，Vmax为213pμ/m in/mg微粒体蛋白。添加b5使Km降至27μm，但对Vmax无影响。这些结果表明，P4502e1是导致肝微粒体中NDMA脱甲基酶和NMBzA脱苄酶活性降低的原因，并且这些活性受细胞色素b5的影响。
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 b5and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates:N-nitrosodimethylamine (NDMA),N-nitrosomethylbenzylamine (NMBzA),p-nitrophenol, phenol, and acetaminophen. ApparentKmandVmaxvalues for NDMA demethylation were 22 μmand 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK 143 cells, which do not contain b5, displayedKmandVmaxvalues of 31 μmand 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5into TK−143 microsomes increased theVmax2.2-fold and decreased theKmto 22 μm. Addition of b5to Hep G2 microsomes resulted in a 1.6-fold increase inVmax, but showed no effect on theKm. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with aKmof 47 μmandVmaxof 213 pmol/min/mg microsomal protein. Addition of b5lowered theKmto 27 μm, but had no effect onVmax. These results demonstrate conclusively that P450 2E1 is responsible for the lowKmforms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.