CLONING, EXPRESSION, AND FUNCTIONAL-CHARACTERIZATION OF RAPID AND SLOW ACETYLATOR POLYMORPHIC N-ACETYLTRANSFERASE ENCODING GENES OF THE SYRIAN-HAMSTER

Syrian hamster acetylation capacity is catalysed by two N-acetyltransf erase isozymes (NAT1 and NAT2). Hamster NAT2 (polymorphic) displays ac etylator-genotype dependent activity resulting in high, intermediate, and low activity levels in homozygous rapid, heterozygous and homozygo us slow acetylators, respectively. A lambda gt10 size-selected genomic library was constructed from Eco RI-digested homozygous slow acetylat or Bio, 82.73/H-Pat(s) congenic hamster DNA and screened with a hamste r NAT1 probe. A 4.2 kb Eco RI insert from a positive clone was subclon ed into PUC18 and the intron-free NAT2 coding region was sequenced. Th e NAT2 coding regions from genomic templates of other homozygous rapid and slow acetylator congenic and inbred hamster lines were amplified by the polymerase chain reaction, cloned, and sequenced. Two NAT2 alle les were found, one (NAT215) from each homozygous rapid acetylator li ne and one (NAT216A) from each homozygous slow acetylator line, NAT2* 15 contained an 870 bp open reading frame encoding a 290 amino acid pr otein. NAT216A was similar except for two silent ((TC)-C-36 and A(633 )G) and one nonsense ((CT)-T-727) substitutions yielding a 242 amino a cid open reading frame. The NAT215 and NAT2*16A alleles were expresse d in Escherichia coli JM105 and the recombinant proteins were characte rized. Electrophoretic mobilities of the NAT2 15 and NAT2 16A recombin ant hamster proteins differed and correlated with the theoretical mole cular weights calculated from their respective open reading frames. NA T2 16A exhibited 500-to 1000-fold lower maximum velocities compared to NAT2 15 for N-acetylation of all arylamine and hydrazine substrates t ested, NAT2 16A also catalysed the metabolic activation of N-hydroxyar ylamines and N-hydroxyarylamides at rates 33- and 23-fold lower than N AT2 15. Intrinsic clearance (V-max/K-m) calculations suggest that N-ac etylation of p-aminobenzoic acid and 2-aminofluorene in Syrian hamster s is catalysed primarily by NAT2 (NAT2 15) in rapid acetylators but by NAT1 (NAT1 9) in slow acetylators. These results provide a molecular basis for rapid and slow acetylator phenotype in the Syrian hamster.