| 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:
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PMID: 1835459 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
Authors:
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J M Staddon; M M Bouzyk; E Rozengurt |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: The Journal of cell biology Volume: 115 ISSN: 0021-9525 ISO Abbreviation: J. Cell Biol. Publication Date: 1991 Nov |
Date Detail:
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Created Date: 1991-12-27 Completed Date: 1991-12-27 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0375356 Medline TA: J Cell Biol Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 949-58 Citation Subset: IM |
Affiliation:
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Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom. |
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| MeSH Terms | |
Descriptor/Qualifier:
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3T3 Cells Adenosine Diphosphate Ribose / metabolism Animals Bacterial Proteins* Bacterial Toxins / metabolism* Cholera Toxin / metabolism Electrophoresis, Polyacrylamide Gel GTP-Binding Proteins / metabolism Kinetics Membrane Proteins / metabolism Mice Niacinamide / pharmacology Pasteurella multocida* Pertussis Toxin Poly(ADP-ribose) Polymerases / metabolism* Solubility Virulence Factors, Bordetella / metabolism |
| Chemical | |
Reg. No./Substance:
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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 2.4.2.30/Poly(ADP-ribose) Polymerases; EC 2.4.2.31/Pertussis Toxin; EC 3.6.1.-/GTP-Binding Proteins |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
| Full Text | |
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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 Download PDF  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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