Reeves P T, Kinnear B F, Minchin R F, Ilett K F
Department of Pharmacology, University of Western Australia, Nedlands.
Mol Pharmacol. 1991 Jan;39(1):42-8.
An immunological evaluation of N-acetyltransferase (NAT) (EC 2.3.1.5) in liver, duodenum, lung, and kidney of the rabbit is described. Polyclonal antibodies to hepatic NAT isolated from rapid acetylator rabbits were raised in a goat and utilized for immunoblot analyses and enzyme inhibition studies. Immunoblot analyses demonstrated that hepatic and duodenal cytosols from rapid but not slow acetylator rabbits contained an immunoreactive 33-kDa protein. No immunoreactivity was observed for lung or kidney cytosols from either rapid or slow acetylators. The inhibition of sulfamethazine and p-aminobenzoic acid acetylation by polyclonal antibodies was investigated using cytosols from rapid and slow acetylator rabbits. With rapid acetylator cytosols, maximal inhibition of hepatic, duodenal, and lung NAT activities was 94.4 +/- 9.0%, 92.5 +/- 8.5%, and 28.3 +/- 2.4%, respectively, for sulfamethazine (500 mM) acetylation and 90.1 +/- 8.0%, 80.2 +/- 6.4%, and 26.7 +/- 3.1%, respectively, for p-aminobenzoic acid (500 microM) acetylation. Using 25 microM p-aminobenzoic acid as substrate, maximal inhibition of NAT activity was 32.0 +/- 2.1% with liver cytosol and 5.8 +/- 0.16% with duodenal cytosol, whereas no inhibition of lung NAT activity was observed. Kidney NAT activity was not inhibited by the polyclonal antibodies. With slow acetylator cytosols, no inhibition of NAT activities was observed. It is concluded that at least two NATs are present in liver, duodenum, and lung of rapid acetylator rabbits. Furthermore, the principal NAT in liver and duodenum is immunologically related to the minor form of lung NAT and is antigenically distinct from kidney NAT of rapid acetylators. Hepatic, duodenal, lung, and kidney NAT(s) of slow acetylator rabbits is (are) immunologically distinct from the major hepatic NAT in rapid acetylators. The data support the model in which the hepatic polymorphism in rabbits is caused by the total lack of the major rapid acetylator hepatic NAT in the phenotypic slow acetylator animal. These observations may have significant implications in the organ-specific toxicities of carcinogens that undergo metabolic activation via N-acetylation.
本文描述了对兔肝脏、十二指肠、肺和肾脏中N-乙酰基转移酶（NAT）（EC 2.3.1.5）的免疫学评估。从快速乙酰化兔中分离出的肝脏NAT的多克隆抗体在山羊体内产生，并用于免疫印迹分析和酶抑制研究。免疫印迹分析表明，快速乙酰化兔而非慢速乙酰化兔的肝脏和十二指肠胞质溶胶中含有一种免疫反应性33 kDa蛋白。快速或慢速乙酰化兔的肺或肾脏胞质溶胶均未观察到免疫反应性。使用快速和慢速乙酰化兔的胞质溶胶研究了多克隆抗体对磺胺二甲嘧啶和对氨基苯甲酸乙酰化的抑制作用。对于快速乙酰化兔的胞质溶胶，在磺胺二甲嘧啶（500 mM）乙酰化反应中，肝脏、十二指肠和肺NAT活性的最大抑制率分别为94.4±9.0%、92.5±8.5%和28.3±2.4%，在对氨基苯甲酸（500 μM）乙酰化反应中分别为90.1±8.0%、80.2±6.4%和26.7±3.1%。以25 μM对氨基苯甲酸为底物，肝脏胞质溶胶对NAT活性的最大抑制率为32.0±2.1%，十二指肠胞质溶胶为5.8±0.16%，而肺NAT活性未观察到抑制作用。肾脏NAT活性未被多克隆抗体抑制。对于慢速乙酰化兔的胞质溶胶，未观察到NAT活性的抑制作用。结论是，快速乙酰化兔的肝脏、十二指肠和肺中至少存在两种NAT。此外，肝脏和十二指肠中的主要NAT与肺NAT的次要形式在免疫学上相关，且在抗原性上与快速乙酰化兔的肾脏NAT不同。慢速乙酰化兔的肝脏、十二指肠、肺和肾脏NAT在免疫学上与快速乙酰化兔的主要肝脏NAT不同。这些数据支持了这样一种模型，即兔肝脏中的多态性是由于表型慢速乙酰化动物完全缺乏主要的快速乙酰化肝脏NAT所致。这些观察结果可能对通过N-乙酰化进行代谢活化的致癌物的器官特异性毒性具有重要意义。
