Activation of Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of RhoA by K+ in a Mg2+ -dependent manner.
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-32P]NAD, incorporation of radioactivity into the recombinant RhoA increased in the presence of KCl. The increase in ADP-ribose incorporation into RhoA due to KCl appeared in the presence of MgCl2 and was abolished by EDTA. C3 was stabilized by KCl, but the stabilization was also seen with BSA. The KCl-induced increase in the ADP-ribosylation was observed even in the presence of BSA during the modification reaction, thus the effect of KCl was not due to the stabilization of C3. While the initial rate of the reaction was increased by KCl, maximum incorporation of ADP-ribose per GST-RhoA molecule did not increase in the presence of KCl. Kinetic analysis revealed that KCl increased Vmax but did not alter Km 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.
Miyaoka
T
T
Departments of Biochemistry and Psychiatry, Shimane Medical University, Izumo.
Tsuchiya
M
M
Hara
N
N
Ishino
H
H
Shimoyama
M
M
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D001483:Base Sequence; D001905:Botulinum Toxins; D003014:Clostridium botulinum / Q000201:enzymology; D004795:Enzyme Stability; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009244:NAD+ Nucleosidase / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011189:Potassium Chloride / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D012266:Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D005982:Glutathione Transferase; D009244:NAD+ Nucleosidase; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D008274:Magnesium; D011188:Potassium