ACTIVATION OF CLOSTRIDIUM-BOTULINUM C3 EXOENZYME-CATALYZED ADP-RIBOSYLATION OF RHOA BY K-DEPENDENT MANNER( IN A MG2+)

The effect of KCl on ADP-ribosylation of the recombinant RhoA protein catalyzed by the Clostridium botulinum C3 enzyme was studied. When the recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was incubated with C3 and [adenylate-P-32]NAD, incorporation of radioa ctivity into the recombinant RhoA increased in the presence of KCl. Th e increase in ADP-ribose incorporation into RhoA due to KCl appeared i n the presence of MgCl2 and was abolished by EDTA. C3 was stabilized b y KCl, but the stabilization was also seen with BSA. The KCl-induced i ncrease in the ADP-ribosylation was observed even in the presence of B SA during the modification reaction, thus the effect of KCl was not du e to the stabilization of C3. While the initial rate of the reaction w as increased by KCl, maximum incorporation of ADP-ribose per GST-RhoA molecule did not increase in the presence of KCl. Kinetic analysis rev ealed that KCl increased V-max but did not alter K-m for either NAD or RhoA. The NAD glycohydrolase activity of C3 was also increased by KCl . These results indicate that KCl directly activates the C3 enzyme.