Document Detail

A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin.
MedLine Citation:
PMID:  16647035     Owner:  NLM     Status:  MEDLINE    
Pseudomonas exotoxin A (PE) inhibits protein synthesis by NAD-dependent ADP-ribosylation of eukaryotic elongation factor 2. Traditionally, toxin activity has been characterized, either in living cells or cell-free systems, using radioactive compounds for quantification. The increased costs of radioactive waste disposal together with heightened security concerns have made the use of radioactive isotopes less attractive for routine laboratory assays. We therefore adapted a cell-free rabbit reticulocyte in vitro transcription-translation system that utilizes a reporter (beta-galactosidase) to measure toxin activity. The assay for PE is rapid, scalable, log-linear, NAD dependent and can be used to assess the neutralizing activity of anti-PE antibody preparations.
Diana V Pastrana; David J FitzGerald
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Intramural     Date:  2006-04-17
Journal Detail:
Title:  Analytical biochemistry     Volume:  353     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-05-29     Completed Date:  2006-08-21     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  266-71     Citation Subset:  IM    
Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, MD 20892-4263, USA.
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MeSH Terms
Adenosine Diphosphate Ribose / metabolism
Bacterial Proteins / immunology,  pharmacology*
Cell-Free System*
Enzyme Activation / drug effects
Genes, Reporter
NAD / metabolism
Peptide Elongation Factor 2 / metabolism
Protein Biosynthesis / drug effects
Protein Synthesis Inhibitors / immunology,  pharmacology*
Transcription, Genetic
beta-Galactosidase / antagonists & inhibitors,  biosynthesis
Reg. No./Substance:
0/Bacterial Proteins; 0/Peptide Elongation Factor 2; 0/Protein Synthesis Inhibitors; 0/pseudomonas exoprotein A protein, Pseudomonas aeruginosa; 20762-30-5/Adenosine Diphosphate Ribose; 53-84-9/NAD; EC

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