Document Detail


Over-expression, purification, and characterization of recombinant human arylamine N-acetyltransferase 1.
MedLine Citation:
PMID:  16003948     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo.
Authors:
Haiqing Wang; Gregory M Vath; Akane Kawamura; Caleb A Bates; Edith Sim; Patrick E Hanna; Carston R Wagner
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The protein journal     Volume:  24     ISSN:  1572-3887     ISO Abbreviation:  Protein J.     Publication Date:  2005 Feb 
Date Detail:
Created Date:  2005-07-11     Completed Date:  2005-11-01     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  101212092     Medline TA:  Protein J     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  65-77     Citation Subset:  IM    
Affiliation:
Department of Medicinal Chemistry University of Minnesota, 308 Harvard Street S.E., Minneapolis, MN 55455, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Arylamine N-Acetyltransferase / antagonists & inhibitors,  genetics*,  isolation & purification,  metabolism
Base Sequence
Chromatography, DEAE-Cellulose
Chromatography, High Pressure Liquid
Cloning, Molecular
Cricetinae
DNA Primers
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics
Folic Acid / analogs & derivatives,  pharmacology
Humans
Plasmids
Recombinant Proteins / antagonists & inhibitors,  genetics,  isolation & purification,  metabolism
Spectrometry, Mass, Electrospray Ionization
Substrate Specificity
Grant Support
ID/Acronym/Agency:
CA55334/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; 0/Recombinant Proteins; 59-30-3/Folic Acid; EC 2.3.1.5/Arylamine N-Acetyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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