Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins.
Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.
Roerig
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Department of Pharmacology, University of Minnesota, Minneapolis 55455.
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005910:Glioma; D006822:Hybrid Cells; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism*; D009447:Neuroblastoma; D009536:Niacinamide / Q000494:pharmacology; D037342:Pertussis Toxin; D010761:Phosphorus Radioisotopes; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins