Effects of four chemicals on N-acetyltransferase activities in human U937 cell line.
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The effects of p-aminobenzoic acid (PABA), procainamide (PA), anisidine (AN) and isoniazid (INH) on N-acetyltransferase (NAT) activities in cultured human cells were determined. PABA increased the specific activity of PABA NAT in the U937 cells but not in the Hep G2 cells. The enzyme activity in the PABA-treated U937 cells was restored to normal within 4 d after removing PABA from medium. These results imply that the PABA NAT activity in the U937 cells can be induced by PABA and the PABA NAT in the U937 cells is different from that in the Hep G2 cells. INH increased the INH NAT specific activity in the U937 cells but decreased the PABA NAT activity. AN decreased both the AN NAT and the PABA NAT specific activities in the U937 cells. PA did not affect the specific activities of PABA NAT or glucose-6-phosphate dihydrogenase (G-6-P DH) in the U937 cells. PABA also increased the specific activities of AN NAT and G-6-P DH. This implies that the induction effect of PABA on the PABA NAT activity is not specific. In this study the PABA NAT specific activity was increased only by PABA, and the INH NAT activity only by INH. However, the AN NAT activity could be induced by PABA but not by AN. These results indicate that induction of some but not all NAT activities has a limiting specificity.Keywords:
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The expression of arylamine N-acetyltransferase (NAT) in leukocytes was investigated using p-aminobenzoic acid (PABA) and sulfamethazine (SMZ), substrates which are preferentially acetylated by the monomorphic NAT1 and polymorphic NAT2 enzymes, respectively. Activity towards both substrates was detected in mononuclear leukocytes (MNL; preparation containing approximately 80% lymphocytes), monocytes and neutrophils. PABA and SMZ acetylation rates were highly correlated in each of the isolated cell types. The NAT in leukocytes displayed a much higher affinity and turnover rate for the acetylation of PABA than for SMZ. These kinetic characteristics suggest that the acetylating activity in human leukocytes is predominantly attributable to the monomorphic enzyme NAT1. Neutrophils showed evidence of biphasic kinetics for SMZ which would indicate the coexpression of NAT1 and low levels of the polymorphic enzyme, NAT2. NAT activity in MNL was not influenced by the acetylator phenotype of the individual. There was, however, a significant correlation between NAT activity in MNL and the in vivo acetylation (urinary metabolite ratio) of p-aminosalicylic acid, which is monomorphically acetylated in humans. The expression of NAT1 in leukocytes and the virtuall absence of NAT2 may have important toxicological implications. The in vitro/in vivo correlation suggests that leukocytes may be a useful marker of systemic NAT1 activity.
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Arylamine N-acetyltransferase (NAT) activity was identified and partially characterized in the bovine lens. According to size-exclusion HPLC, the molecular mass of the arylamine NAT is approximately 30-kDa. Based upon substrate specificity analysis, it is best described as an arylamine NAT which has some ability to N-acetylate arylalkylamines. This arylamine NAT acetylates para-aminobenzoic acid thereby demonstrating a monomorphic pattern of N-acetylation. It demonstrates low sensitivity to methotrexate inhibition as indicated by the relatively high IC50 value (470 microM). NAT could be involved in lenticular detoxification of both endogenous amines and exogenous drugs.
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The effects of p-aminobenzoic acid (PABA), procainamide (PA), anisidine (AN) and isoniazid (INH) on N-acetyltransferase (NAT) activities in cultured human cells were determined. PABA increased the specific activity of PABA NAT in the U937 cells but not in the Hep G2 cells. The enzyme activity in the PABA-treated U937 cells was restored to normal within 4 d after removing PABA from medium. These results imply that the PABA NAT activity in the U937 cells can be induced by PABA and the PABA NAT in the U937 cells is different from that in the Hep G2 cells. INH increased the INH NAT specific activity in the U937 cells but decreased the PABA NAT activity. AN decreased both the AN NAT and the PABA NAT specific activities in the U937 cells. PA did not affect the specific activities of PABA NAT or glucose-6-phosphate dihydrogenase (G-6-P DH) in the U937 cells. PABA also increased the specific activities of AN NAT and G-6-P DH. This implies that the induction effect of PABA on the PABA NAT activity is not specific. In this study the PABA NAT specific activity was increased only by PABA, and the INH NAT activity only by INH. However, the AN NAT activity could be induced by PABA but not by AN. These results indicate that induction of some but not all NAT activities has a limiting specificity.
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Abstract This unit describes methods for measuring the activity of arylamine N‐acetyltransferases (NAT). Genetic polymorphisms in NAT 1 and NAT 2 have been associated with susceptibility to aromatic amines carcinogens and effects of therapeutic drugs. Evaluation of the activities associated with substrates of NATs is helpful in elucidating the contribution of these enzymes to the pharmacologic and toxicologic effects of arylamines and hydrazines.
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N-Acetylation plays an important role in the metabolism of a wide variety of hydrazine drugs and arylamine drugs and carcinogens. Humans have genetically determined differences in their N-acetyltransferase activities and are phenotypically classified as rapid or slow acetylators. Mice have a similar genetic polymorphism in N-acetyltransferase activity and have been used as models of the human polymorphism in many studies of the toxicology and carcinogenicity of arylamines. Recently, two N-acetyltransferase genes, Nat-1 and Nat-2, were cloned from rapid (C57BL/6J) and slow (A/J) acetylator mouse strains. The genomic clone encoding NAT-1 is identical in rapid and slow acetylator mouse strains, whereas the clone encoding NAT-2 differs between rapid and slow strains by a single base pair, which changes the encoded amino acid from Asn99 in the rapid acetylator strain to Ile99 in the slow acetylator strain. In this report, the N-acetylation polymorphism in mice was investigated by transiently expressing the cloned N-acetyltransferase genes in COS-1 cells. The intronless coding regions of Nat-1 and Nat-2 showed different substrate specificities; isoniazid was a preferred substrate for NAT-1, whereas p-aminobenzoic acid was preferred for NAT-2(99asn) and NAT-2(99ile). All three enzymes acetylated 2-aminofluorene, but none of them acetylated sulfamethazine. Kinetic constants determined for the expressed enzymes with 2-aminofluorene and p-aminobenzoic acid indicated that Km values were not significantly different between the enzymes, although the Vmax value of NAT-2(99asn) was consistently 2-3-fold higher than that of NAT-1 or NAT-2(99ile). Nat-1 and Nat-2 encoded mRNAs of approximately 1.4 kilobases in livers of rapid and slow acetylators. Nat-2 mRNA was more abundant in liver than Nat-1 mRNA. The abundance of Nat-2 mRNA and Nat-1 mRNA was equivalent in both rapid and slow acetylator mouse strain livers. Incubation of transfected COS-1 cell cytosols at 37 degrees showed that the time for decline of NAT activity to 50% of its initial value was 45 hr for NAT-1, 60 hr for NAT-2(99asn), and 4 hr for NAT-2(99ile). This 15-fold difference in the heat stability of the rapid and slow isoforms of NAT activity was also observed in cytosols from rapid and slow acetylator livers. Comparison of the rates of translation of the rapid and slow isoforms of NAT-2 in an in vitro system showed that NAT-2(99asn) was translated at approximately twice the rate of NAT-2(99ile).(ABSTRACT TRUNCATED AT 400 WORDS)
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Arylamine N-acetyltransferase
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Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.
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Arylamine N-acetyltransferase
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