Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes.
A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT; EC 2.3.1.5) enzyme activity divides populations into distinguishable "slow acetylator" and "rapid acetylator" phenotypes. Two human genes, NAT1 and NAT2, encoding NAT proteins [DNA Cell Biol. 9:193-203 (1990)] were transiently expressed in cultured monkey kidney COS-1 cells, and the resulting recombinant NAT1 and NAT2 proteins were compared with N-acetyltransferase activities in human liver cytosol with respect to their stability, chromatographic behavior on anion exchange columns, electrophoretic mobility, and arylamine acceptor substrate specificity. NAT1 was far less stable in vitro than NAT2. Under conditions designed to optimize enzyme stability, anion exchange chromatography experiments revealed that enzymes corresponding to both recombinant NAT1 and NAT2 were expressed in human liver. Recombinant and human liver NAT1 enzymes showed the same characteristic selectivity (low apparent Km, high Vmax) for the "monomorphic" substrates p-aminosalicylic acid and p-aminobenzoic acid. Such substrates fail to discriminate between the acetylator phenotypes in vivo. The same criteria established that recombinant NAT2 was indistinguishable from one of two previously observed N-acetyltransferases (NAT2A and NAT2B) whose liver contents correlate with acetylator phenotype in human populations. Recombinant NAT2 and the liver NAT2 isoforms NAT2A and NAT2B selectivity N-acetylated the "polymorphic" substrates sulfamethazine and procainamide, whose disposition in vivo is affected by the acetylation polymorphism. Interestingly, the carcinogen 2-aminofluorene was very efficiently metabolized by both NAT1 and NAT2. Independent regulation of NAT1 and NAT2 genes was suggested by a lack of correlation of NAT1 and NAT2 enzyme activities in cytosols from 39 human livers. The results provide strong evidence that the NAT2 locus is the site of the human acetylation polymorphism. In addition, the use of recombinant NAT1 and NAT2 will allow us to predict whether any given arylamine will be polymorphically acetylated in humans.
Grant
D M
DM
Department of Pharmacology, University of Basel, Switzerland.
Blum
M
M
Beer
M
M
Meyer
U A
UA
D000107:Acetylation; D000818:Animals; D000838:Anions; D001191:Arylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004586:Electrophoresis; D004795:Enzyme Stability; D005449:Fluorenes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D011110:Polymorphism, Genetic / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity
D000838:Anions; D002273:Carcinogens; D005449:Fluorenes; D007527:Isoenzymes; D011994:Recombinant Proteins; C012177:2-aminofluorene; D001191:Arylamine N-Acetyltransferase