Narimatsu Shizuo, Oda Maiko, Hichiya Hiroyuki, Isobe Takashi, Asaoka Kazuo, Hanioka Nobumitsu, Yamano Shigeru, Shinoda Sumio, Yamamoto Shigeo
Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Chem Biol Interact. 2005 Feb 28;152(1):1-12. doi: 10.1016/j.cbi.2005.01.006.
A cDNA encoding a novel cytochrome P450 1A2 (CYP1A2) was cloned from the liver of an adult female Japanese monkey. The CYP1A2 protein was expressed in yeast cells and its enzymatic properties were compared with those of marmoset CYP1A2 using ethoxyresorufin (ER) and phenacetin (PN) as substrates. The nucleotide sequence of Japanese monkey CYP1A2 revealed 94.7, 99.5 and 93.5% identities to those of human, cynomolgus monkey and marmoset monkey CYP1A2, respectively. Multiple amino acid sequence alignment of Japanese monkey CYP1A2 with CYP1A2 of humans, cynomolgus monkeys and marmosets showed that Japanese monkey CYP1A2 had 92.4, 99.0 and 91.9% identities to the human, cynomolgus monkey and marmoset enzymes, respectively. Kinetic studies demonstrated that the enzymatic properties as ER and PN O-deethylases were considerably different between the Japanese monkey and the marmoset CYP1A2. Furthermore, both of these reactions in liver microsomal fractions from the Japanese monkey and marmoset showed biphasic kinetics. On the basis of the kinetic parameters, it is suggested that Japanese monkey CYP1A2 is a high-K(m) enzyme in both ER and PN O-deethylations, whereas marmoset CYP1A2 is a high-K(m) and low-K(m) enzyme in ER and PN O-deethylations, respectively. alpha-Naphthoflavone, an inhibitor of human CYP1A1 and CYP1A2, did not completely inhibit the liver microsomal oxidations of ER and PN even at the highest concentration (50muM), supporting the notion that CYP1A2 enzymes are not the sole ER or PN O-deethylase in Japanese monkey and marmoset liver microsomes. Inhibitory effects of furafylline, an inhibitor of human CYP1A2, on ER O-deethylation by recombinant CYP1A2 enzymes were much lower than those of alpha-naphthoflavone, but marmoset CYP1A2 was more sensitive to furafylline than Japanese monkey CYP1A2. These results indicate that the properties of Japanese monkey CYP1A2 are considerably different from those of marmoset CYP1A2.
从成年雌性日本猕猴肝脏中克隆出一种编码新型细胞色素P450 1A2（CYP1A2）的cDNA。将CYP1A2蛋白在酵母细胞中表达，并以乙氧香豆素（ER）和非那西丁（PN）为底物，将其酶学性质与狨猴CYP1A2的酶学性质进行比较。日本猕猴CYP1A2的核苷酸序列与人、食蟹猴和狨猴CYP1A2的核苷酸序列分别具有94.7%、99.5%和93.5%的同一性。日本猕猴CYP1A2与人、食蟹猴和狨猴的CYP1A2的多氨基酸序列比对显示，日本猕猴CYP1A2与人、食蟹猴和狨猴的酶分别具有92.4%、99.0%和91.9%的同一性。动力学研究表明，日本猕猴和狨猴CYP1A2作为ER和PN O-脱乙基酶的酶学性质有很大差异。此外，日本猕猴和狨猴肝脏微粒体组分中的这两种反应均呈现双相动力学。根据动力学参数，提示日本猕猴CYP1A2在ER和PN O-脱乙基反应中均为高K(m)酶，而狨猴CYP1A2在ER和PN O-脱乙基反应中分别为高K(m)酶和低K(m)酶。α-萘黄酮是人CYP1A1和CYP1A2的抑制剂，即使在最高浓度（50μM）时也不能完全抑制ER和PN的肝脏微粒体氧化，这支持了CYP1A2酶不是日本猕猴和狨猴肝脏微粒体中唯一的ER或PN O-脱乙基酶的观点。人CYP1A2的抑制剂呋拉茶碱对重组CYP1A2酶催化的ER O-脱乙基反应的抑制作用远低于α-萘黄酮，但狨猴CYP1A2比日本猕猴CYP1A2对呋拉茶碱更敏感。这些结果表明，日本猕猴CYP1A2的性质与狨猴CYP1A2的性质有很大不同。
