caa a22 4500 397555784 CHVBK 20180308164801.0 cr unu---uuuuu 161202e199601 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021209 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021209 Activation of Clostridium botulinum C3 Exoenzyme-Catalyzed ADP-Ribosylation of RhoA by K+in a Mg2+-Dependent Manner [Elektronische Daten] [Tsuyoshi Miyaoka, Mikako Tsuchiya, Nobumasa Hara, Hiroshi Ishino, Makoto Shimoyama] 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 KC1. 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 KC1 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. © by the Journal of Biochemistry Regular Papers nationallicence activation nationallicence ADP-ribosylation nationallicence Clostridium botulinum C3 exoenzyme nationallicence potassium ion nationallicence recombinant GST-RhoA fusion protein nationallicence Miyaoka Tsuyoshi Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut Tsuchiya Mikako Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut Hara Nobumasa Central Research Laboratories, Shimane Medical University Izumo, Shimane 693 aut Ishino Hiroshi Psychiatry, Shimane Medical University Izumo, Shimane 693 aut Shimoyama Makoto Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut The Journal of Biochemistry Oxford University Press 119/1(1996-01), 200-207 0021-924X 119:1<200 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021209 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021209 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Miyaoka Tsuyoshi Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut NATIONALLICENCE 700 1- Tsuchiya Mikako Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut NATIONALLICENCE 700 1- Hara Nobumasa Central Research Laboratories, Shimane Medical University Izumo, Shimane 693 aut NATIONALLICENCE 700 1- Ishino Hiroshi Psychiatry, Shimane Medical University Izumo, Shimane 693 aut NATIONALLICENCE 700 1- Shimoyama Makoto Departments of Biochemistry, Shimane Medical University Izumo, Shimane 693 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/1(1996-01), 200-207 0021-924X 119:1<200 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford