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A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.
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MedLine Citation:
PMID:  1835459     Owner:  NLM     Status:  MEDLINE    
Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.
J M Staddon; M M Bouzyk; E Rozengurt
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Journal of cell biology     Volume:  115     ISSN:  0021-9525     ISO Abbreviation:  J. Cell Biol.     Publication Date:  1991 Nov 
Date Detail:
Created Date:  1991-12-27     Completed Date:  1991-12-27     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  0375356     Medline TA:  J Cell Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  949-58     Citation Subset:  IM    
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
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MeSH Terms
3T3 Cells
Adenosine Diphosphate Ribose / metabolism
Bacterial Proteins*
Bacterial Toxins / metabolism*
Cholera Toxin / metabolism
Electrophoresis, Polyacrylamide Gel
GTP-Binding Proteins / metabolism
Membrane Proteins / metabolism
Niacinamide / pharmacology
Pasteurella multocida*
Pertussis Toxin
Poly(ADP-ribose) Polymerases / metabolism*
Virulence Factors, Bordetella / metabolism
Reg. No./Substance:
0/Bacterial Proteins; 0/Bacterial Toxins; 0/Membrane Proteins; 0/Pasteurella multocida toxin; 0/Virulence Factors, Bordetella; 20762-30-5/Adenosine Diphosphate Ribose; 9012-63-9/Cholera Toxin; 98-92-0/Niacinamide; EC Polymerases; EC Toxin; EC 3.6.1.-/GTP-Binding Proteins

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

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Journal Information
Journal ID (nlm-ta): J Cell Biol
Journal ID (publisher-id): J. Cell Biol.
ISSN: 0021-9525
ISSN: 1540-8140
Publisher: The Rockefeller University Press
Article Information
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Print publication date: Day: 2 Month: 11 Year: 1991
Volume: 115 Issue: 4
First Page: 949 Last Page: 958
ID: 2289951
Publisher Id: 92064654
PubMed Id: 1835459

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

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