Fukuta H, Ohi H, Uchida T, Komori M, Kitada M, Kamataki T
Division of Analytical Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Mutat Res. 1992 Sep;269(1):97-105. doi: 10.1016/0027-5107(92)90164-w.
A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.
一种编码比格犬细胞色素P - 450（Dah1）形式的互补DNA（cDNA）被插入酵母表达载体pAAH5的乙醇脱氢酶（ADH）启动子和终止子区域之间，该cDNA是大鼠、小鼠和人类CYP1A1 cDNA的直系同源物。导入所得重组质粒pDC - 1后，酿酒酵母菌株AH22细胞每个细胞合成多达1.5×10⁵个细胞色素P - 450蛋白分子（P - 450（Dah1））。P - 450（Dah1）的一氧化碳结合还原形式在447nm处显示吸收峰，P - 450（Dah1）的比含量约为每毫克微粒体蛋白0.1纳摩尔P - 450。P - 450（Dah1）与大鼠P - 448 - H（CYP1A2）和犬P - 450 - D2（CYP1A2）的抗体发生交叉反应。在umu试验中，P - 450（Dah1）最有效地激活2 - 氨基 - 3 - 甲基 - 咪唑[4,5 - f]喹啉（IQ）和2 - 氨基 - 3,4 - 二甲基咪唑[4,5 - f]喹啉（MeIQ），并且对苯并[a]芘表现出高活性的芳烃羟化酶活性。
