Abstract
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.
Highlights
Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic metabolizing enzymes with an essential role in the metabolism of many drugs and carcinogens[1]
In order to assess the crossmRNA expression of N-acetyltransferase 2 (NAT2) in UV5/N-acetyltransferase 1 (NAT1) cells, and NAT1 expression in UV5/NAT2 cells, respectively, the expression of both proteins was evaluated in both cell lines
No detectable expression of NAT2 was found in UV5/ NAT1 cells (Fig. 1b); no expression of NAT1 was detected on UV5/NAT2 cells (Fig. 1c)
Summary
Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic metabolizing enzymes with an essential role in the metabolism of many drugs and carcinogens[1]. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) share 87% and 81% of sequence homology at the mRNA and protein level, respectively Both proteins have 290 amino acids with a molecular mass of 33.9 and 33.6 kDa, respectively. Despite their high similarity, NAT1 and NAT2 exhibit different substrate specificity and tissue distribution. The basis of interindividual variability in the NAT2 acetylation polymorphism were first described in tuberculosis patients being treated with isoniazid[6] This finding gained relevance with the discovery that many hydrazines and arylamine drugs and carcinogens were affected by the same genetic polymorphism, having an effect on the toxicity, therapeutic efficiency or carcinogenesis risk of such compounds[7,8]. A previous report in human liver biopsies reported a decrease in the amount of immunodetectable NAT2 protein in subjects with a slow acetylation phenotype[14], the present work focused on www.nature.com/scientificreports the quantification of NAT2 protein expression in cryoplateable human hepatocytes and its relationship to NAT2 genotype and NAT2 catalytic activity
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