Tsyrlov I B, Goldfarb I S, Gelboin H V
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Arch Biochem Biophys. 1993 Dec;307(2):259-66. doi: 10.1006/abbi.1993.1588.
Kinetics of benzo[alpha]pyrene hydroxylase (AHH), 7-methoxyresorufin o-demethylase (MROD), and 7-ethoxyresorufin o-deethylase (EROD) were estimated in microsomes of Hep G2 cells infected with a recombinant vaccinia virus bearing mouse CYP1A1 or CYP1A2 cDNAs. The kcat and Km values obtained were compared with those of liver microsomal and purified mouse CYP1A1 and CYP1A2. In the matter of AHH activity, the kcat CYP1A1/CYP1A2 ratios were 21.2, 12.3, and 1.5 for expressed, microsomal, and purified CYPs, respectively. As to MROD activity, the kcat CYP1A2/CYP1A1 ratio was 3.0 for both expressed and microsomal CYPs and was 8.0 for purified CYPs. As regards EROD activity, the kcat CYP1A2/CYP1A1 ratios were 1.0, 1.1, and 6.25 for expressed, microsomal, and purified CYPs, respectively. Whereas furafylline displayed an isozyme-specific inhibition of CYP1A2-catalyzing MROD and EROD activities, alpha-naphthoflavone was an equally strong inhibitor of AHH activity of the CYP1A1s and MROD activities of the CYP1A2s. Immunodepleted polyclonal anti-CYP1A1(-A2) and anti-CYP1A2(-A1) showed an isozyme-specific immunoblotting and inhibition of mouse CYP1A1 and CYP1A2 while monoclonal antibody (Mab) 1-7-1 displayed a striking difference between its immunoblotting and inhibitory effects. Western blot/densitometry analysis revealed a 4.8 times lower binding of Mab 1-7-1 to cDNA-expressed CYP1A2 than to CYP1A1. The results demonstrate the reliability of the vaccinia virus expression system for studies on the enzymology of mouse CYP1A1 and CYP1A2.
在感染携带小鼠CYP1A1或CYP1A2 cDNA的重组痘苗病毒的Hep G2细胞微粒体中，对苯并[a]芘羟化酶（AHH）、7-甲氧基试卤灵O-脱甲基酶（MROD）和7-乙氧基试卤灵O-脱乙基酶（EROD）的动力学进行了评估。将获得的kcat和Km值与肝微粒体以及纯化的小鼠CYP1A1和CYP1A2的kcat和Km值进行比较。就AHH活性而言，表达的、微粒体的和纯化的细胞色素P450（CYP）的kcat CYP1A1/CYP1A2比率分别为21.2、12.3和1.5。至于MROD活性，表达的和微粒体的CYP的kcat CYP1A2/CYP1A1比率均为3.0，纯化的CYP的该比率为8.0。关于EROD活性，表达的、微粒体的和纯化的CYP的kcat CYP1A2/CYP1A1比率分别为1.0、1.1和6.25。虽然呋拉茶碱对CYP1A2催化的MROD和EROD活性表现出同工酶特异性抑制，但α-萘黄酮对CYP1A1的AHH活性和CYP1A2的MROD活性具有同样强的抑制作用。免疫耗尽的多克隆抗CYP1A1（-A2）和抗CYP1A2（-A1）显示出对小鼠CYP1A1和CYP1A2的同工酶特异性免疫印迹和抑制作用，而单克隆抗体（Mab）1-7-1在其免疫印迹和抑制作用之间表现出显著差异。蛋白质免疫印迹/光密度分析显示，Mab 1-7-1与cDNA表达的CYP1A2的结合比与CYP1A1的结合低4.8倍。结果证明了痘苗病毒表达系统在小鼠CYP1A1和CYP1A2酶学研究中的可靠性。
