Cloned mouse N-acetyltransferases: enzymatic properties of expressed Nat-1 and Nat-2 gene products.
Cloned mouse N-acetyltransferases: enzymatic properties of expressed Nat-1 and Nat-2 gene products.
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发表时间：
1992-08
影响因子：
3.6
通讯作者：
K. Martell;G. Levy;W. Weber
中科院分区：
文献类型：
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作者：
K. Martell;G. Levy;W. Weber
N-乙酰化在多种肼类药物、芳胺类药物和致癌物的代谢中起着重要作用。人类在其N-乙酰转移酶活性方面具有遗传决定的差异，并且在表型上被分类为快速乙酰化者或缓慢乙酰化者。小鼠在N-乙酰基转移酶活性方面具有相似的遗传多态性，并已在芳香胺的毒理学和致癌性的许多研究中用作人类多态性的模型。最近，从快乙酰化小鼠（C57 BL/6 J）和慢乙酰化小鼠（A/J）中克隆了两个N-乙酰转移酶基因，Nat-1和Nat-2。编码NAT-1的基因组克隆在快速乙酰化小鼠品系和慢速乙酰化小鼠品系中是相同的，而编码NAT-2的克隆在快速乙酰化小鼠品系和慢速乙酰化小鼠品系之间的差异在于单个碱基对，这将编码的氨基酸从快速乙酰化小鼠品系中的Asn 99改变为慢速乙酰化小鼠品系中的Ile 99。本研究通过在COS-1细胞中瞬时表达克隆的N-乙酰基转移酶基因来研究小鼠的N-乙酰化多态性。Nat-1和Nat-2的内含子编码区显示出不同的底物特异性;异烟肼是NAT-1的优选底物，而对氨基苯甲酸是NAT-2（99 asn）和NAT-2（99 ile）的优选底物。这三种酶都能乙酰化2-氨基芴，但都不能乙酰化磺胺二甲嘧啶。用2-氨基芴和对氨基苯甲酸测定的表达酶的动力学常数表明，尽管NAT-2（99 asn）的Vmax值始终比NAT-1或NAT-2（99 ile）高2-3倍，但酶之间的Km值没有显著差异。Nat-1和Nat-2在快乙酰化和慢乙酰化的肝脏中编码约1.4种乙酰化酶的mRNA。Nat-2 mRNA在肝脏中的表达量高于Nat-1 mRNA。Nat-2 mRNA和Nat-1 mRNA的丰度在快速乙酰化和缓慢乙酰化小鼠品系的肝脏中是相等的。将转染的COS-1细胞胞质溶胶在37 ℃下孵育，NAT活性下降到其初始值的50%的时间对于NAT-1为45小时，对于NAT-2（99 asn）为60小时，对于NAT-2（99 ile）为4小时。这15倍的差异，在热稳定性的快速和慢速亚型的NAT活性也观察到在细胞溶胶从快速和慢速乙酰化肝脏。在体外系统中比较NAT-2的快速和慢速同种型的翻译速率表明，NAT-2（99 asn）的翻译速率大约是NAT-2（99 ile）的两倍。（400字处截断摘要）
N-Acetylation plays an important role in the metabolism of a wide variety of hydrazine drugs and arylamine drugs and carcinogens. Humans have genetically determined differences in their N-acetyltransferase activities and are phenotypically classified as rapid or slow acetylators. Mice have a similar genetic polymorphism in N-acetyltransferase activity and have been used as models of the human polymorphism in many studies of the toxicology and carcinogenicity of arylamines. Recently, two N-acetyltransferase genes, Nat-1 and Nat-2, were cloned from rapid (C57BL/6J) and slow (A/J) acetylator mouse strains. The genomic clone encoding NAT-1 is identical in rapid and slow acetylator mouse strains, whereas the clone encoding NAT-2 differs between rapid and slow strains by a single base pair, which changes the encoded amino acid from Asn99 in the rapid acetylator strain to Ile99 in the slow acetylator strain. In this report, the N-acetylation polymorphism in mice was investigated by transiently expressing the cloned N-acetyltransferase genes in COS-1 cells. The intronless coding regions of Nat-1 and Nat-2 showed different substrate specificities; isoniazid was a preferred substrate for NAT-1, whereas p-aminobenzoic acid was preferred for NAT-2(99asn) and NAT-2(99ile). All three enzymes acetylated 2-aminofluorene, but none of them acetylated sulfamethazine. Kinetic constants determined for the expressed enzymes with 2-aminofluorene and p-aminobenzoic acid indicated that Km values were not significantly different between the enzymes, although the Vmax value of NAT-2(99asn) was consistently 2-3-fold higher than that of NAT-1 or NAT-2(99ile). Nat-1 and Nat-2 encoded mRNAs of approximately 1.4 kilobases in livers of rapid and slow acetylators. Nat-2 mRNA was more abundant in liver than Nat-1 mRNA. The abundance of Nat-2 mRNA and Nat-1 mRNA was equivalent in both rapid and slow acetylator mouse strain livers. Incubation of transfected COS-1 cell cytosols at 37 degrees showed that the time for decline of NAT activity to 50% of its initial value was 45 hr for NAT-1, 60 hr for NAT-2(99asn), and 4 hr for NAT-2(99ile). This 15-fold difference in the heat stability of the rapid and slow isoforms of NAT activity was also observed in cytosols from rapid and slow acetylator livers. Comparison of the rates of translation of the rapid and slow isoforms of NAT-2 in an in vitro system showed that NAT-2(99asn) was translated at approximately twice the rate of NAT-2(99ile).(ABSTRACT TRUNCATED AT 400 WORDS)