Ferguson R J, Doll M A, Rustan T D, Baumstark B R, Hein D W
Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks 58202-9037.
Pharmacogenetics. 1994 Apr;4(2):82-90.
N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.
N-乙酰基转移酶在芳胺和肼类药物及致癌物的代谢中起重要作用。人类的N-乙酰化表型可能使个体易患多种药物和外源性物质诱导的毒性反应及致癌作用。叙利亚仓鼠表达两种N-乙酰基转移酶同工酶；一种随乙酰化基因型而变化（多态性），被称为NAT2；另一种则不变化（单态性），被称为NAT1。通过聚合酶链反应从纯合快速乙酰化和纯合缓慢乙酰化的近交系和近交仓鼠基因组DNA模板中克隆了无内含子的NAT1编码区并进行了测序。来自纯合快速（NAT1）和纯合缓慢（NAT1s）乙酰化仓鼠的NAT1等位基因在一个核苷酸上存在差异，但该突变是沉默的，推导的氨基酸序列没有变化。为了表征NAT1等位基因的酶产物，我们开发了一种原核表达系统。通过表达盒聚合酶链反应扩增NAT1r和NAT1s等位基因，并将其亚克隆到基于tac启动子的质粒载体pKK223-3中，以便在大肠杆菌JM105菌株中过量表达重组NAT1。从选定的插入NAT1的转化体诱导培养物中产生了高水平的能够进行N-乙酰化、O-乙酰化和N,O-乙酰化的可溶性蛋白质。重组NAT1r和NAT1s蛋白在底物特异性、比活性、米氏动力学性质、内在稳定性和电泳迁移率方面没有差异。此外，过表达的NAT1蛋白表现出从叙利亚仓鼠肝脏和结肠胞质溶胶中分离出的NAT1的底物特异性和电泳迁移率特征。
