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首页> 外文期刊>Xenobiotica: the fate of foreign compounds in biological systems >Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.
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Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

机译：人细胞色素P4501A1（CYP1A1）变体和人NADPH-细胞色素P450还原酶在杆状病毒/昆虫细胞系统中的共表达。

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1.Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.
机译：1，三种人细胞色素P4501A1（CYP1A1）变体，野生型（CYP1A1.1），CYP1A1.2（1462V）和CYP1A1.4（T461N）在拟南芥中与人NADPH-P450还原酶（OR）共表达（Sf9）昆虫细胞通过杆状病毒共感染来构建一个合适的系统，用于研究CYPA1多态性在外源和内源底物代谢中的作用。 2.检查了一系列条件，以针对诸如感染的相对多重性（MOI），收获时间，血红素前体补充和翻译后稳定等参数优化共表达。对于所有CYP1A1变体，达到了火种优化的条件，几乎相同的表达水平和OR / CYP1A1摩尔比（20：1）。 3.从共感染细胞中分离出的微粒体表现出乙氧基异佛手菌素脱羧酶活性（nmol / min（-1）nmol（-1）CYP1A1）分别为16.0（CYP1A1.1），20.5（CYP1A1.2）和22.5（CYP1A1.4）。用所有酶变体将戊氧基间苯二酚脱烷基的速度慢约10-20倍。 4.所有三种CYP1A1变体均具有代谢致癌物前苯并[a] py的活性，其中野生型酶显示最高活性，其次是CYP1A1.4（60％）和CYP1A1.2（40 ％）。每个变体产生所有主要代谢产物，包括最终致癌物质的前体B [a] P-7,8-二氢二醇。 5.这些研究表明，杆状病毒介导的通过共感染共表达的所有CYP1A1变体均产生具有类似摩尔OR / CYP1A1摩尔比的功能活性酶系统，从而提供了合适的前提条件，以通过不同的CY1A1变体。

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