Syrian hamster monomorphic N-acetyltransferase (NAT1) alleles: amplification, cloning, sequencing, and expression in E. coli.
N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.
Ferguson
R J
RJ
Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks 58202-9037.
Doll
M A
MA
Rustan
T D
TD
Baumstark
B R
BR
Hein
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005449:Fluorenes / Q000378:metabolism; D006881:Hydroxyacetylaminofluorene / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008647:Mesocricetus / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA
D005449:Fluorenes; D007527:Isoenzymes; D011994:Recombinant Proteins; D006881:Hydroxyacetylaminofluorene; C001368:N-hydroxy-2-aminofluorene; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1