A Novel Approach to Detect Toxin-Catalyzed ADP-Ribosylation in Intact Cells: Its Use to Study the Action of Pasteurella multocida Toxin

• Published in: The Journal of cell biology Vol. 115; no. 4; pp. 949 - 958
• Main Authors: Staddon, James M., Bouzyk, Mark M., Rozengurt, Enrique
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.11.1991; The Rockefeller University Press
• Subjects: 3T3 Cells; Adenosine Diphosphate Ribose - metabolism; Animals; Bacterial Proteins; Bacterial Toxins - metabolism; Bacteriology; Biological and medical sciences; Cell membranes; Cells; Cholera; Cholera Toxin - metabolism; Cultured cells; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Gels; GTP-Binding Proteins - metabolism; Kinetics; Membrane Proteins - metabolism; Mice; Microbiology; Niacinamide - pharmacology; Pasteurella multocida; Pertussis Toxin; Poly(ADP-ribose) Polymerases - metabolism; Radioactive decay; Solubility; Swiss 3T3 cells; Toxins; Virulence Factors, Bordetella - metabolism; Whooping cough; Pasteurellaceae; Toxin; Enzymatic activity; Enzyme; ADP-ribosyltransferase; Bacteria; Pasteurella multocida
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.115.4.949

Certain microbial toxins are ADP-ribosyl-transferases, 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 fluorography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein in intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95°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.