A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.
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.
Staddon
J M
JM
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Bouzyk
M M
MM
Rozengurt
E
E
D016475:3T3 Cells; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009536:Niacinamide / Q000494:pharmacology; D016979:Pasteurella multocida; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D012995:Solubility; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D008565:Membrane Proteins; C062258:Pasteurella multocida toxin; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins