title: Purification of hepatic polymorphic arylamine N-acetyltransferase from homozygous rapid acetylator inbred hamster: identity with polymorphic N-hydroxyarylamine-O-acetyltransferase.
abstract: The polymorphic acetyltransferase isozyme expressed in homozygous rapid acetylator inbred hamster liver cytosol was purified over 2000-fold by sequential Q-Sepharose fast-flow anion-exchange chromatography, Sephacryl S-200 high-resolution size-exclusion chromatography, Mono Q anion-exchange fast-protein liquid chromatography, and preparative polyacrylamide gel electrophoresis. The isozyme migrated as a single homogeneous monomer following both preparative and sodium dodecyl sulfate-polyacrylamide electrophoresis. The molecular weight was estimated at 34,170 following elution via size-exclusion chromatography and 35,467 following migration via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneous polymorphic acetyltransferase exhibited a broad substrate specificity; it catalyzed the acetyl coenzyme A-dependent N-acetylation of p-aminobenzoic acid, carbocyclic arylamine carcinogens such as 2-aminofluorene, 4-aminobiphenyl and beta-naphthylamine, and heterocyclic arylamine carcinogens such as 2-aminodipyrido[1,2-a:3'2'd]imidazole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. It also readily catalyzed the acetyl coenzyme A-dependent metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to DNA adducts but not the metabolic activation (via intramolecular, N,O-acetyltransfer) of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl to DNA adducts. Conversely, the partially purified monomorphic acetyltransferase isozyme from the same hamsters readily catalyzed the metabolic activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl, and rates of metabolic activation of these substrates did not differ between homozygous rapid and slow acetylator liver, intestine, kidney, and lung cytosols. Heat inactivation rates for the purified polymorphic acetyltransferase isozyme were first order and indistinguishable for the acetyl coenzyme A-dependent N-acetylation and O-acetylation activities. The results strongly suggest the expression of a single polymorphic acetyltransferase product of the hamster polymorphic acetyltransferase gene that catalyzes both acetyl coenzyme A-dependent N-acetylation and O-acetylation of arylamine and N-hydroxyarylamine carcinogens but not the metabolic activation of N-hydroxy-N-acetylarylamines (arylhydroxamic acids) via intramolecular N,O-acetyltransfer. Consequently, acetylator genotype-dependent metabolic activation of N-hydroxyarylamines to a DNA adduct in hamster is catalyzed by direct O-acetylation of the hydroxyl group and not via sequential N-acetylation followed by N,O-acetyltransfer.
authors: Department of Pharmacology, Morehouse School of Medicine, Atlanta, Georgia 30310.
authors: A
authors: A
authors: Trinidad
authors: D W
authors: DW
authors: Hein
authors: T D
authors: TD
authors: Rustan
authors: R J
authors: RJ
authors: Ferguson
authors: L S
authors: LS
authors: Miller
authors: K D
authors: KD
authors: Bucher
authors: W G
authors: WG
authors: Kirlin
authors: F
authors: F
authors: Ogolla
authors: A F
authors: AF
authors: Andrews
mesh_terms: D000123:Acetyltransferases; D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006720:Homozygote; D007422:Intestines / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008168:Lung / Q000201:enzymology*; D008297:Male; D008647:Mesocricetus; D008970:Molecular Weight; D011110:Polymorphism, Genetic
chemical_list: D004247:DNA; D000217:Acyltransferases; D000123:Acetyltransferases; C019736:N-hydroxyarylamine O-acetyltransferase; D001191:Arylamine N-Acetyltransferase