title: A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, MD 20892-4263, USA.
authors.forename: Diana V
authors.initials: DV
authors.lastname: Pastrana
authors.forename: David J
authors.initials: DJ
authors.lastname: FitzGerald
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000494:pharmacology*; D002474:Cell-Free System; D004789:Enzyme Activation / Q000187:drug effects; D017930:Genes, Reporter; D009243:NAD / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000276:immunology / Q000494:pharmacology*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D020652:Peptide Elongation Factor 2; D011500:Protein Synthesis Inhibitors; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D001616:beta-Galactosidase