Cloning, expression, and functional characterization of rapid and slow acetylator polymorphic N-acetyl-transferase encoding genes of the Syrian hamster.
Cloning, expression, and functional characterization of rapid and slow acetylator polymorphic N-acetyl-transferase encoding genes of the Syrian hamster.
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DOI：
10.1097/00008571-199602000-00004
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发表时间：
1996-02
期刊：
影响因子：
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通讯作者：
R. J. Ferguson;M. Doll;T. Rustan;David W. Hein
中科院分区：
文献类型：
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作者：
R. J. Ferguson;M. Doll;T. Rustan;David W. Hein
叙利亚仓鼠乙酰化能力由两种N-乙酰转移酶同工酶（NAT 1和NAT 2）催化。NANAT 2（多态性）显示乙酰化基因型依赖性活性，分别导致纯合快速、杂合和纯合慢速乙酰化基因的高、中和低活性水平。λ gt 10大小选择的基因组文库由EcoRI消化的纯合慢乙酰化因子Bio构建。82.73/H-Pats同源仓鼠DNA，并用仓鼠NAT 1探针筛选。从阳性克隆中插入一个4.2kb的EcoRI插入片段，亚克隆到pUC 18中，并对无内含子的NAT 2编码区进行测序。通过聚合酶链反应扩增来自其他纯合的快速和缓慢乙酰化同源和近交仓鼠品系的基因组模板的NAT 2编码区，克隆并测序。发现两个NAT 2等位基因，一个（NAT 2 *15）来自每个纯合快速乙酰化株系，一个（NAT 2 * 16 A）来自每个纯合缓慢乙酰化株系。NAT 2 *15含有一个870 bp的开放阅读框，编码290个氨基酸。NAT 2 * 16 A除了两个沉默（T36 C和A633 G）和一个无义（C727 T）取代外是相似的，产生242个氨基酸的开放阅读框架。将NAT 2 *15和NAT 2 * 16 A基因在大肠杆菌JM 105中表达，并对重组蛋白进行了鉴定。NAT 2 15和NAT 2 16 A重组仓鼠蛋白的电泳迁移率不同，并与从其各自的开放阅读框计算的理论分子量相关。NAT 2 16 A表现出500- 1000倍低的最大速度相比，NAT 2 15的N-乙酰化的所有芳胺和肼底物测试。NAT 2 16 A还催化N-羟基芳胺和N-羟基芳酰胺的代谢活化，速率比NAT 2 15低33倍和23倍。固有清除率（Vmax/Km）的计算表明，N-乙酰化的对氨基苯甲酸和2-氨基芴在叙利亚仓鼠是催化主要由NAT 2（NAT 2 15）在快速乙酰化，但由NAT 1（NAT 1 9）在缓慢乙酰化。这些结果为叙利亚仓鼠的快乙酰化和慢乙酰化表型提供了分子基础。
Syrian hamster acetylation capacity is catalysed by two N-acetyltransferase isozymes (NAT1 and NAT2). Hamster NAT2 (polymorphic) displays acetylator-genotype dependent activity resulting in high, intermediate, and low activity levels in homozygous rapid, heterozygous and homozygous slow acetylators, respectively. A lambda gt10 size-selected genomic library was constructed from Eco RI-digested homozygous slow acetylator Bio. 82.73/H-Pats congenic hamster DNA and screened with a hamster NAT1 probe. A 4.2 kb Eco RI insert from a positive clone was subcloned into pUC18 and the intron-free NAT2 coding region was sequenced. The NAT2 coding regions from genomic templates of other homozygous rapid and slow acetylator congenic and inbred hamster lines were amplified by the polymerase chain reaction, cloned, and sequenced. Two NAT2 alleles were found, one (NAT2*15) from each homozygous rapid acetylator line and one (NAT2*16A) from each homozygous slow acetylator line. NAT2*15 contained an 870 bp open reading frame encoding a 290 amino acid protein. NAT2*16A was similar except for two silent (T36C and A633G) and one nonsense (C727T) substitutions yielding a 242 amino acid open reading frame. The NAT2*15 and NAT2*16A alleles were expressed in Escherichia coli JM105 and the recombinant proteins were characterized. Electrophoretic mobilities of the NAT2 15 and NAT2 16A recombinant hamster proteins differed and correlated with the theoretical molecular weights calculated from their respective open reading frames. NAT2 16A exhibited 500-to 1000-fold lower maximum velocities compared to NAT2 15 for N-acetylation of all arylamine and hydrazine substrates tested. NAT2 16A also catalysed the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides at rates 33- and 23-fold lower than NAT2 15. Intrinsic clearance (Vmax/Km) calculations suggest that N-acetylation of p-aminobenzoic acid and 2-aminofluorene in Syrian hamsters is catalysed primarily by NAT2 (NAT2 15) in rapid acetylators but by NAT1 (NAT1 9) in slow acetylators. These results provide a molecular basis for rapid and slow acetylator phenotype in the Syrian hamster.