CLONING, SEQUENCING, AND RECOMBINANT EXPRESSION OF NAT1, NAT2, AND NAT3 DERIVED FROM THE C3H HEJ (RAPID) AND A/HEJ (SLOW) ACETYLATOR INBREDMOUSE - FUNCTIONAL-CHARACTERIZATION OF THE ACTIVATION AND DEACTIVATION OF AROMATIC AMINE CARCINOGENS/

An acetylator polymorphism has been described in the mouse and the inb red strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, r espectively. The NAT1, NAT2, and NAT3 genes from C3H/HeJ and A/HeJ ace tylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced. They w ere then subcloned into the prokaryotic expression vector pKK223-3 and expressed in Escherichia coli strain JM105. The 870-bp nucleotide cod ing region of NAT1 and NAT3 did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouse N AT1 and NAT3 sequences. However, NAT2 did differ between the rapid and slow acetylator strains with an A(296) T transition which causes a (A sn(99) --> Ile) substitution in the deduced amino acid sequence. Recom binant NAT1, NAT2, and NAT3 proteins catalyzed N-, O-, and N,O-acetylt ransferase activities. NAT3 catalyzed aromatic amine N-acetyltransfera se activities at very low rates, which confirms a previous study. Appa rent K-m and V-max kinetic constants for N-acetylation were 5- to 10-f old lower for recombinant mouse NAT1 than NAT2. Intrinsic clearances f or recombinant mouse NAT1- and NAT2-catalyzed N-acetylation of aromati c amine carcinogens were comparable. Both recombinant mouse NAT1 and N AT2 catalyzed the metabolic activation of N-hydroxyarylamine (O-acetyl ation) and N-hydroxyarylamide (N,O-acetylation) carcinogens. Recombina nt mouse NAT3 catalyzed N,O-acetylation at very low rates, while O-ace tylation was undetectable. No difference was observed between rapid an d slow acetylator recombinant NAT2 proteins to activate aromatic amine s by O- or N,O-acetylation, in substrate specificity, expression of im munoreactive protein, electrophoretic mobility, or N-acetyltransferase Michaelis-Menten kinetic constants. However, the slow acetylator reco mbinant NAT2 protein was over 10-fold less stable than rapid acetylato r recombinant NAT2. These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2 and NAT3 proteins and co nfirm and extend previous studies on the molecular basis for the acety lation polymorphism in the mouse. (C) 1997 Academic Press.