SYRIAN-HAMSTER MONOMORPHIC N-ACETYLTRANSFERASE (NAT1) ALLELES - AMPLIFICATION, CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI

N-acetyltransferases have an important role in the metabolism of aryla mine and hydrazine drugs an carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-in duced toxicities and carcinogenesis. Syrian hamsters express two N-ace tyltransferase isozymes; one varies with acetylator genotype (polymorp hic) and has been termed NAT2; the other does not (monomorphic) and ha s been termed NAT1. The intronless NAT1 coding region was cloned via t he polymerase chain reaction from homozygous rapid acetylator and homo zygous slow acetylator congenic and inbred hamster genomic DNA templat es and sequenced. The NAT1 alleles from the homozygous rapid (NAT1r) a nd homozygous slow (NAT1s) acetylator hamsters differed in one nucleot ide, but the mutation is silent with no change in deduced amino acid s equence. To characterize the enzyme products of the NAT1 alleles, we d eveloped a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and su bcloned into the tac promoter-based plasmid vector pKK223-3 for over-p roduction of recombinant NAT1 in E. coli strain JM105. induced culture s from selected NAT1-inserted transformants yielded high levels of sol uble protein capable of N-acetylation, O-acetylation, and N,O-acetylat ion. The recombinant NAT1r and NAT1s proteins did not differ in substr ate specificity, specific activity, Michaelis-Menten kinetic propertie s, intrinsic stability, and electrophoretic mobility. Also, the over-e xpressed NAT1 proteins displayed substrate-specificity and electrophor etic mobilities characteristic of NAT1 isolated from Syrian hamster li ver and colon cytosols.