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Research ArticleArticle

Characterization of Naturally Occurring and Recombinant HumanN-Acetyltransferase Variants Encoded byNAT1*

Jesús H. de León, Kostas P. Vatsis and Wendell W. Weber
Molecular Pharmacology August 2000, 58 (2) 288-299; DOI: https://doi.org/10.1124/mol.58.2.288
Jesús H. de León
Department of Pharmacology, Medical School, The University of Michigan, Ann Arbor, Michigan
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Kostas P. Vatsis
Department of Pharmacology, Medical School, The University of Michigan, Ann Arbor, Michigan
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Wendell W. Weber
Department of Pharmacology, Medical School, The University of Michigan, Ann Arbor, Michigan
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Abstract

The genotype at the NAT1* locus of an interethnic population of 38 unrelated subjects was determined by direct sequencing of 1.6-kb fragments amplified by PCR. The coding exon alone and together with the 3′ noncoding exon of the wild-type (NAT1*4) and the three mutant alleles (NAT1*10, *11, and *16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT1*4/*4 and NAT1*10/*10homozygotes was also isolated. Recombinant and leukocyte cytosolic preparations were thoroughly characterized byN-acetylation activity with several NAT1-specific and -selective substrates, as well as by steady-state kinetics with varying amounts of the substrate (fixed acetyl CoA) and acetyl CoA (fixed substrate), thermodynamics, stability, and protein immunoreactivity with a polyclonal human anti-NAT1. The polyadenylation signal mutation in the 3′ noncoding sequence of NAT1*10 affected none of the aforementioned parameters evaluated both with recombinantNAT1*10 and with the naturally occurring allele. Function was also unaffected by the coding and 3′ noncoding exon mutations in NAT1*11. In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3′ untranslated region ofNAT1*16 ostensibly caused disruption of the predicted secondary structure of the pre-mRNA for NAT1 16, culminating in parallel 2-fold decreases in the amount and catalytic activity of NAT1 16 in COS-1 cell cytosol. This novel finding inN-acetylation pharmacogenetics clearly demonstrates a direct link between reduced catalytic activity and structural alteration in the 3′ untranslated region of an NATvariant (NAT1*16) brought about by mutation.

Footnotes

    • Received November 17, 1999.
    • Accepted March 8, 2000.
  • Send reprint requests to: Dr. Kostas P. Vatsis, Department of Biological Chemistry, The University of Michigan Medical School, M5440 Medical Science Building I, Ann Arbor, MI 48109-0606. E-mail:kvatsis{at}umich.edu

  • ↵1 Present address: Health Effects Laboratory Division, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, WV 26505-2845.

  • ↵2 Present address: Department of Biological Chemistry, The University of Michigan Medical School, M5440 Medical Science Building I, Ann Arbor, MI 48109-0606.

  • This work was supported by Grants GM44965 and CA39018 from the National Institutes of Health. The data are taken from a thesis submitted by Jesús H. de León to the Rackham School of Graduate Studies, The University of Michigan, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology. Preliminary reports of this study have been presented at the annual meetings of the American Society of Human Genetics [Am J Hum Genet 55(suppl):A340, 1994] and of Experimental Biology (FASEB J 10:A456, 1996), as well as at the North American meeting of the International Society for the Study of Xenobiotics (ISSX Proc 12:97, 1997).

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Molecular Pharmacology: 58 (2)
Molecular Pharmacology
Vol. 58, Issue 2
1 Aug 2000
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Research ArticleArticle

Characterization of Naturally Occurring and Recombinant HumanN-Acetyltransferase Variants Encoded byNAT1*

Jesús H. de León, Kostas P. Vatsis and Wendell W. Weber
Molecular Pharmacology August 1, 2000, 58 (2) 288-299; DOI: https://doi.org/10.1124/mol.58.2.288

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Research ArticleArticle

Characterization of Naturally Occurring and Recombinant HumanN-Acetyltransferase Variants Encoded byNAT1*

Jesús H. de León, Kostas P. Vatsis and Wendell W. Weber
Molecular Pharmacology August 1, 2000, 58 (2) 288-299; DOI: https://doi.org/10.1124/mol.58.2.288
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