Cloning, expression, and functional characterization of two mutant (NAT2(191) and NAT2(341/803)) and wild-type human polymorphic N-acetyltransferase (NAT2) alleles.
Cloning, expression, and functional characterization of two mutant (NAT2(191) and NAT2(341/803)) and wild-type human polymorphic N-acetyltransferase (NAT2) alleles.
复制标题
DOI：
--
复制
发表时间：
1994-05
期刊：
影响因子：
--
通讯作者：
R. J. Ferguson;M. Doll;T. Rustan;K. Gray;D. Hein
中科院分区：
文献类型：
--
作者：
R. J. Ferguson;M. Doll;T. Rustan;K. Gray;D. Hein
N-乙酰化多态性通过NAT 2基因座的单基因遗传将个体分离为快速、中间和缓慢乙酰化表型。在以前的研究（Arch. Toxicol. 67，445-452，1993），我们发现了基于聚合酶链反应-限制性片段长度多态性分析的表观NAT 2乙酰化基因型、体外结肠芳胺N-乙酰转移酶活性和非裔美国人中慢乙酰化表型的预期频率之间的差异，这表明存在尚未确定的突变NAT 2等位基因。通过对NAT 2基因的PCR产物进行克隆和测序，发现了两个新的NAT 2等位基因。一个等位基因（NAT 2（191））在核苷酸191处含有点突变[G-->A（Arg-->Gln）]，而另一个等位基因（NAT 2（341/803））含有两个点突变[341 T-->C（Ile-->Thr）; 803 A-->G（Lys-->Arg）]。在原核表达系统中表达两个突变的NAT 2和NAT 2 wt等位基因。NAT 2（191）和NAT 2（341/803）突变等位基因均表达功能性N-乙酰转移酶，能够催化芳胺N-乙酰化和N-羟基-2-氨基芴的代谢活化（通过O-乙酰化）。然而，NAT 2（191）和NAT 2（341/803）各自表现出显著较低的N-和0-乙酰化能力，并且本质上比NAT 2 wt.
The N-acetylation polymorphism segregates individuals into rapid, intermediate, and slow acetylator phenotypes via monogenic inheritance at the NAT2 locus. In a previous study (Arch. Toxicol. 67, 445-452, 1993), we uncovered discrepancies between apparent NAT2 acetylator genotype based on polymerase chain reaction-restriction fragment length polymorphism analysis, in vitro colon arylamine N-acetyltransferase activity, and expected frequency of slow acetylator phenotype in African-Americans, which suggested the presence of not yet defined mutant NAT2 alleles. Two novel NAT2 alleles were discovered after cloning and sequencing of NAT2 polymerase chain reaction products. One allele (NAT2(191)) contained a point mutation at nucleotide 191 [G-->A (Arg-->Gln)], whereas the other allele (NAT2(341/803)) contained two point mutations [341T-->C (Ile-->Thr); 803A-->G (Lys-->Arg)]. The two mutant NAT2 and the NAT2wt alleles were expressed in a prokaryotic expression system. Both the NAT2(191) and NAT2(341/803) mutant alleles expressed functional N-acetyltransferases capable of catalyzing both arylamine N-acetylation and the metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene. However, the NAT2(191) and NAT2(341/803) each exhibited significantly lower N- and O-acetylation capacity and were intrinsically less stable than NAT2wt.