
<ArticleTitle>Co-expression of human <prot>cytochrome P4501A1</prot> (<prot>CYP1A1</prot>) variants and human <prot>NADPH-cytochrome P450 reductase</prot> in the baculovirus/insect cell system.</ArticleTitle>
<AbstractText>1.Three human <prot>cytochrome P4501A1</prot> (<prot>CYP1A1</prot>) variants, wild-type (<prot>CYP1A1.1</prot>), <prot>CYP1A1.2</prot> (1462V) and <prot>CYP1A1.4</prot> (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/<prot>CYP1A1</prot> ratios (20:1) were attained for all <prot>CYP1A1</prot> variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) <prot>CYP1A1</prot>) of 16.0 (<prot>CYP1A1.1</prot>), 20.5 (<prot>CYP1A1.2</prot>) and 22.5 (<prot>CYP1A1.4</prot>). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three <prot>CYP1A1</prot> variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by <prot>CYP1A1.4</prot> (60%) and <prot>CYP1A1.2</prot> (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 <prot>CYP1A1</prot> variants yields functionally active enzyme systems with similar molar OR/<prot>CYP1A1</prot> ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different <prot>CY1A1</prot> variants.</AbstractText>
