Abstract
Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze both the N-acetylation and O-acetylation of arylamines and N-hydroxyarylamines. Humans possess two functional N-acetyltransferase genes, NAT1 and NAT2, as well as a nonfunctional pseudogene, NATP. Previous studies have identified Nat1 and Nat2 genes in the rat. In this study, we identified and characterized a third rat N-acetyltransferase gene (Nat3) consisting of a single open reading frame of 870 base pairs encoding a 290-amino acid protein, analogous to the previously identified human and rat N-acetyltransferase genes. Rat Nat3 nucleotide sequence was 77.2 and 75.9% identical to human NAT1 and NAT2, respectively. Rat Nat3 amino acid sequence was 68.6 and 67.2% identical to human NAT1 and NAT2, respectively. Rat Nat1, Nat2, and Nat3 were each cloned and recombinantly expressed in Escherichia coli. Recombinant rat Nat3 exhibited thermostability intermediate between recombinant rat Nat1 and Nat2. Recombinant rat Nat3 was functional and catalyzed the N-acetylation of several arylamine substrates, including 3-ethylaniline, 3,5-dimethylaniline, 5-aminosalicylic acid, 4-aminobiphenyl, 4,4′-methylenedianiline, 4,4′-methylenebis(2-chloroaniline), and 2-aminofluorene, and the O-acetylation of N-hydroxy-4-aminobiphenyl. The relative affinities of arylamine carcinogens such as 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and 2-aminofluorene for N- and O-acetylation via recombinant rat Nat3 were comparable with recombinant rat Nat1 and higher than for recombinant rat Nat2. This study is the first to report a third arylamine N-acetyltransferase isozyme with significant functional capacity.
Footnotes
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This work was partially supported by United States Public Health Service Grant CA34627 from the National Cancer Institute and Training Grant ES011564 from the National Institute of Environmental Health Sciences. A preliminary report of this work was presented at the 95th Annual Meeting of the American Association for Cancer Research; 2004 Mar 27-31; Orlando, FL. American Association for Cancer Research, Philadelphia, PA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.108399.
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ABBREVIATIONS: NAT1, human N-acetyltransferase 1; NAT2, human N-acetyltransferase 2; Nat1, rat or mouse N-acetyltransferase 1; Nat2, rat or mouse N-acetyltransferase 2; Nat3, rat or mouse N-acetyltransferase 3; NATP, human N-acetyltransferase pseudogene; ORF, open reading frame; kb, kilobase(s); BN, Brown Norway; F344, Fisher 344; SPRD, Sprague-Dawley; WKY, Wistar Kyoto; PCR, polymerase chain reaction; 3EA, 3-ethylaniline; 35DMA, 3,5-dimethylaniline; OT, o-toluidine; 26DMA, 2,6-dimethylaniline; 5AS, 5-aminosalicylic acid; PABA, p-aminobenzoic acid; PAA, p-aminoacetanilide; 3A4MAA, 3-amino-4-methoxyacetanilide; pABG, N-(p-aminobenzoyl)glutamate; PA, procainamide; ABP, 4-aminobiphenyl; MDA, 4,4′-methylenedianiline; MOCA, 4,4′-methylenebis(2-chloroaniline); AF, 2-aminofluorene; INH, isoniazid; SMZ, sulfamethazine; N-OH-ABP, N-hydroxy-4-aminobiphenyl; HPLC, high-performance liquid chromatography.
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↵1 This work constitutes partial fulfillment for the Ph.D. program in Pharmacology and Toxicology at the University of Louisville School of Medicine.
- Received May 24, 2006.
- Accepted July 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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