Yang Y H, Miranda C L, Henderson M C, Wang-Buhler J L, Buhler D R
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-7301, USA.
Drug Metab Dispos. 2000 Nov;28(11):1279-83.
LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.
LMC2是在性未成熟的虹鳟鱼（Onchorynchus mykiss）中发现的最丰富的组成型表达的肝细胞色素P450，也是激活致癌物黄曲霉毒素B1（AFB1）的同工酶。这种P450已被克隆、测序，并命名为CYP2K1。本报告描述了具有酶活性的CYP2K1（BV-CYP2K1）在杆状病毒草地贪夜蛾（Sf9）昆虫细胞中的异源表达，以及与先前纯化的LMC2 P450相比其催化和免疫反应特性。表达CYP2K1酶和LMC2的Sf9细胞匀浆在大鼠NADPH-细胞色素P450还原酶存在下均催化月桂酸的羟基化和AFB1的环氧化。LMC2和BV-CYP2K1都主要在（ω-1）位催化月桂酸氧化，在（ω-2）位有少量氧化。当P450孵育混合物中含有AFB1、谷胱甘肽（GSH）以及小鼠肝细胞溶胶或纯化的大鼠GSH转移酶时，AFB1环氧化物-谷胱甘肽共轭物的出现间接表明了AFB1环氧化物的形成。当使用手性柱通过HPLC分析由BV-CYP2K1和纯化的LMC2产生的AFB1环氧化物-GSH共轭物时，其保留时间与由已知仅形成AFB1外环氧物的人P450 CYP3A4产生的保留时间相同。因此，这些结果证实，cDNA表达的CYP2K1蛋白在催化和免疫方面与纯化的鳟鱼LMC2相同，并且这两种酶主要产生AFB1具有高度致癌性的立体异构体外环氧物形式。
