Regular Article
Cloning, Sequencing, and Recombinant Expression ofNAT1, NAT2,andNAT3Derived from the C3H/HeJ (Rapid) and A/HeJ (Slow) Acetylator Inbred Mouse: Functional Characterization of the Activation and Deactivation of Aromatic Amine Carcinogens

https://doi.org/10.1006/taap.1996.8036Get rights and content

Abstract

An acetylator polymorphism has been described in the mouse and the inbred strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, respectively. TheNAT1, NAT2,andNAT3genes from C3H/HeJ and A/HeJ acetylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced. They were then subcloned into the prokaryotic expression vector pKK223-3 and expressed inEscherichia colistrain JM105. The 870-bp nucleotide coding region ofNAT1andNAT3did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouseNAT1andNAT3sequences. However,NAT2did differ between the rapid and slow acetylator strains with an A296T transition which causes a (Asn99→ Ile) substitution in the deduced amino acid sequence. Recombinant NAT1, NAT2, and NAT3 proteins catalyzedN-, O-,andN,O-acetyltransferase activities. NAT3 catalyzed aromatic amineN-acetyltransferase activities at very low rates, which confirms a previous study. ApparentKmandVmaxkinetic constants forN-acetylation were 5- to 10-fold lower for recombinant mouse NAT1 than NAT2. Intrinsic clearances for recombinant mouse NAT1- and NAT2-catalyzedN-acetylation of aromatic amine carcinogens were comparable. Both recombinant mouse NAT1 and NAT2 catalyzed the metabolic activation ofN-hydroxyarylamine (O-acetylation) andN-hydroxyarylamide (N,O-acetylation) carcinogens. Recombinant mouse NAT3 catalyzedN,O-acetylation at very low rates, whileO-acetylation was undetectable. No difference was observed between rapid and slow acetylator recombinant NAT2 proteins to activate aromatic amines byO- orN,O-acetylation, in substrate specificity, expression of immunoreactive protein, electrophoretic mobility, orN-acetyltransferase Michaelis–Menten kinetic constants. However, the slow acetylator recombinant NAT2 protein was over 10-fold less stable than rapid acetylator recombinant NAT2. These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2, and NAT3 proteins and confirm and extend previous studies on the molecular basis for the acetylation polymorphism in the mouse.

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