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 α-subunit of GTPbinding proteins (G-proteins) in the presence of NAD +. Pertussis toxin also decreases the electrophoretic mobility of the a-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 α-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD + (0.026-1.3 μM), there was a linear increase in the formation of the slower migrating α-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 a-subunit. Thus, in addition to PTX, NAD + was required for the mobility shift and the slower migrating a-subunit is likely the ADP-ribosylated form.