Department of Pharmacology & Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky, 40202, USA.
Sci Rep. 2020 May 5;10(1):7566. doi: 10.1038/s41598-020-64508-0.
Human N-acetyltransferases (NAT; EC 2.3.1.5) catalyze the N-acetylation of arylamine and hydrazine drugs and the O-acetylation of N-hydroxylated metabolites of aromatic and heterocyclic amines. Two different isoforms of this protein, N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2), are expressed in human hepatocytes. Both are encoded by a single 870-bp open reading frame that exhibits genetic polymorphisms in human populations. NAT1 and NAT2 share more than 85% gene and protein sequence, making it challenging to produce antibodies with high specificity for NAT1 or NAT2. In the present study, we compared methods for the quantification of immunoreactive NAT1 and NAT2 with seven different antibodies and investigated the relationship of NAT2 genotype to NAT2 mRNA and protein expression in cryopreserved human hepatocytes. Sulfamethazine (NAT2-selective substrate) and NAT2 protein expression differed significantly with NAT2 acetylator genotype (p < 0.0001). NAT2 protein expression and sulfamethazine NAT2 catalytic activity correlated highly across the cryopreserved human hepatocytes of rapid, intermediate, and slow acetylator NAT2 genotypes. In conclusion, our data describe a specific analytical method for the quantification of NAT1 and NAT2 protein expression. We showed that the NAT2 activity in human hepatocytes is directly correlated to expression levels of NAT2 protein but not mRNA.
人类 N-乙酰基转移酶（NAT；EC 2.3.1.5）催化芳基胺和肼类药物的 N-乙酰化，以及芳香族和杂环胺的 N-羟基化代谢物的 O-乙酰化。这种蛋白质的两种不同同工型，N-乙酰基转移酶 1（NAT1）和 N-乙酰基转移酶 2（NAT2），在人肝细胞中表达。两者均由一个 870bp 的开放阅读框编码，该阅读框在人类群体中存在遗传多态性。NAT1 和 NAT2 共享超过 85%的基因和蛋白质序列，因此很难产生针对 NAT1 或 NAT2 的高特异性抗体。在本研究中，我们比较了使用七种不同抗体定量检测免疫反应性 NAT1 和 NAT2 的方法，并研究了 NAT2 基因型与冷冻保存的人肝细胞中 NAT2 mRNA 和蛋白表达的关系。磺胺甲噁唑（NAT2 选择性底物）和 NAT2 蛋白表达在 NAT2 乙酰化酶基因型之间存在显著差异（p<0.0001）。NAT2 蛋白表达和磺胺甲噁唑 NAT2 催化活性在快速、中间和慢乙酰化酶 NAT2 基因型的冷冻保存人肝细胞中高度相关。总之，我们的数据描述了一种用于定量检测 NAT1 和 NAT2 蛋白表达的特定分析方法。我们表明，人肝细胞中的 NAT2 活性与 NAT2 蛋白表达水平直接相关，但与 mRNA 无关。
