Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression

Area of Science:

• Pharmacogenomics
• Molecular Biology
• Biochemistry

Background:

• N-acetylation, catalyzed by hepatic arylamine N-acetyltransferase (NAT), is a key pathway for drug metabolism and xenobiotic detoxification.
• Genetic polymorphism in NAT influences drug response and is of significant clinical importance in human populations.

Purpose of the Study:

• To clone and characterize human arylamine N-acetyltransferase (NAT) genes.
• To identify the gene encoding the polymorphic NAT enzyme responsible for drug metabolism.

Main Methods:

• Cloning of three human NAT genes (NAT1, NAT2, NATP) from leukocyte DNA using rabbit NAT cDNA.
• Gene expression analysis in monkey kidney COS-1 cells following transfection.
• Enzyme activity assays using sulfamethazine substrate.
• Western blot analysis to determine protein molecular weight.
• Amino acid sequence comparison with purified human NAT peptides.

Main Results:

• Two functional human NAT genes, NAT1 and NAT2, were identified, both lacking introns and assigned to chromosome 8.
• Transfected NAT1 and NAT2 expressed functional NAT proteins with distinct molecular weights (33 kD and 31 kD, respectively).
• NAT2's protein product and deduced amino acid sequence strongly suggested it encodes the polymorphic NAT enzyme found in human liver.

Conclusions:

• The human NAT2 gene encodes the polymorphic arylamine N-acetyltransferase enzyme.
• NAT1 also encodes a functional NAT enzyme, contributing to drug metabolism.
• The NATP gene is likely a non-functional pseudogene due to deleterious mutations.