Murakami H, Yabusaki Y, Ohkawa H
DNA. 1986 Feb;5(1):1-10. doi: 10.1089/dna.1986.5.1.
A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S. cerevisiae cells.
采用冈山和伯格（1982年）的方法，从苯巴比妥诱导的大鼠肝脏中制备的多聚腺苷酸RNA（poly(A)RNA）中克隆出大鼠NADPH-细胞色素P-450还原酶的全长cDNA。除了四个单碱基差异外，cDNA和氨基酸序列与波特和卡斯珀（1985年）报道的序列一致。通过将还原酶cDNA插入酵母乙醇脱氢酶I（ADH）启动子和终止子区域之间，构建了三种表达质粒。质粒pARF1和pTRF2在ADH启动子和起始密码子之间的长度略有不同；将它们导入酿酒酵母AH22细胞后，每个细胞分别合成约1×10³和5×10³个还原酶蛋白分子。第三种质粒pARM1，在两个还原酶cDNA表达单元之间含有一个细胞色素P-450MC cDNA表达单元，每个细胞合成4×10⁵个细胞色素P-450MC血红蛋白和1×10⁴个还原酶蛋白分子。同时合成两种大鼠酶的AH22/pARM1菌株的细胞提取物，与仅合成大鼠细胞色素P-450MC的AH22/pAMC1菌株的提取物相比，显示出更高的细胞色素c还原酶活性和细胞色素P-450MC依赖性7-乙氧基香豆素O-脱乙基活性。AH22/pARM1菌株细胞提取物中的7-乙氧基香豆素O-脱乙基活性部分受到抗大鼠还原酶IgG添加的抑制。此外，整个AH22/pARM1细胞对乙酰苯胺和7-乙氧基香豆素的单加氧酶活性高于对照AH22/pAMC1细胞。这些结果表明在酿酒酵母细胞中构建了由大鼠NADPH-细胞色素P-450还原酶和大鼠细胞色素P-450MC组成的功能性电子传递链。
