Trepanier L A, Ray K, Winand N J, Spielberg S P, Cribb A E
Department of Pharmacology, Cornell University, College of Veterinary Medicine, Ithaca, NY 14853, U.S.A.
Biochem Pharmacol. 1997 Jul 1;54(1):73-80. doi: 10.1016/s0006-2952(97)00140-8.
The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.
本研究的目的是确定犬类中胞质N - 乙酰转移酶（NAT）活性异常且绝对缺乏的分子基础，NAT是一种对芳胺和肼类化合物代谢很重要的酶。尽管底物浓度范围从10微摩尔到4毫摩尔，且孵育时间范围很广，但在犬肝脏胞质溶胶中未检测到NAT对两种NAT底物对氨基苯甲酸和磺胺二甲嘧啶的活性。同样，在犬肝脏胞质溶胶的蛋白质免疫印迹分析中，未发现与NAT抗体发生免疫反应的蛋白质。用全长人NAT2 cDNA对总共25只纯种和杂种犬以及8只野生犬科动物的基因组DNA进行Southern印迹分析，结果表明所有犬科动物中均不存在NAT序列。使用针对哺乳动物NAT1和NAT2共有序列设计的简并引物对基因组DNA进行聚合酶链反应扩增，在人、小鼠、兔和猫的DNA中产生了预期大小的产物，但在任何犬或野生犬科动物中均未产生NAT产物。这些结果支持以下结论：家犬胞质NAT缺乏是由于完全不存在NAT基因，并且这种缺陷在其他犬科动物中也存在。
