Naunyn-Schmiedeberg's Arch Pharmacol (1996) 354:693-697 © Springer-Verlag 1996 Peter Sehr • Ingo Just • Klaus Aktories ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity Received: 31 May 1996/Accepted: 12 September 1996 Abstract Effects of ADP-ribosylation of skeletal muscle a-actin by Clostridium perfringens iota toxin and by tur- key erythrocyte ADP-ribosyltransferase A on profilin-regu- lated nucleotide exchange and ATPase activity were com- pared. ADP-ribosylation of actin at Arg177 by Clostridium perfi'ingens iota toxin increased the nucleotide dissociation rate from 2.2x10 -3 s-1 to 4.5x10 -3 s-1 without affecting the profilin-induced stimulation of nucleotide exchange. In contrast, ADP-ribosylation of actin at Arg95/Arg372 in- duced by turkey erythrocyte transferase decreased the nu- cleotide dissociation rate to 1.5×10 .3 s ~ and inhibited the profilin-induced stimulation of nucleotide exchange. Whereas toxin-induced ADP-ribosylation at Arg177 blocked actin ATPase, basal G-actin ATPase was not al- tered by ADP-ribosylation at Arg95/Arg372 but inhibited profilin effects on actin ATPase. Key words ADP-ribosylation - Actin profilin C. perfringens iota toxin • Turkey erythrocyte transferase type A • nucleotide exchange - ATPase Introduction C. botulinum C2 toxin and C. perfringens iota toxin are potent cytotoxins which act on the eukaryotic organism by ADP-ribosylation of actin. The toxins ADP-ribosylate ac- tin selectively at Arg177, a modification that inhibits actin polymerisation most likely by sterical hindrance (Aktories et al. 1986; Schering et al. 1988). Furthermore, ADP-ribo- sylated actin acts like a capping protein to inhibit polymer- isation of unmodified actin at the fast polymerising (barbed) ends of F-actin without affecting interaction of monomeric actin with the slow polymerising (pointed) R Sehr. I. Just- K. Aktories (~) Institut ftir Pharmakologie und Toxikologie der Albert-Ludwigs- Universit~it Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany ends of actin filaments (Wegner and Aktories 1988; Weigt et al. 1989). Moreover, toxin-induced ADP-ribosylation of actin inhibits the nucleation activity of the actin-gelsolin complex (Wille et al. 1992). It has been shown that actin is substrate for ADP-ribo- sylation by several eukaryotic ADP-ribosyltransferases (Fujita et al. 1995; Matsuyama and Tsuyama 1991; Tera- shima et al. 1992) supporting the notion that bacterial tox- ins possibly mimic endogenous regulatory processes. Re- cently, the ADP-ribosyltransferase type A from turkey ery- throcytes has been used as a model for eukaryotic modifi- cation of actin (Just et al. 1995). This enzyme ADP-ribo- sylates actin at Arg95 and Arg372, a modification which delays actin polymerisation (Just et al. 1995). Dynamic changes in the organisation of the actin cyto- skeleton depend on the presence of a variety actin-binding proteins that associate with either F- or G-actin. Profilin, a 12-15 kDa protein, appears to be one of the most impor- tant proteins involved in regulation of actin polymerisation (Theriot and Mitchison 1993). Profilin-actin complexes di- rectly elongate actin filaments at their barbed ends. In the profilin-actin complex, monomeric actin ATPase is inhib- ited (Tobacman and Korn 1982) and the exchange of nu- cleotide (ATP/ADP) bound to actin is largely increased (Mockrin and Korn 1980; Perelroizen et al. 1995). Be- cause ADP-ribosylation of actin largely affects also actin ATPase and nucleotide exchange, we studied the influence of toxin- and transferase A induced ADP-ribosylation of actin on profilin-regulated nucleotide exchange and ATPase activity of actin. Materials and methods Materials. [32P]NAD was obtained from NEN (Bad Homburg, Ger- 1,N ,ethenoadenosine 5'-tri- many), poly-L-proline (MW -5000) and 6 phosphate were from Sigma (Deisenhofen, Germany). YM l0 mem- branes were supplied from Amicon (Dansvers, Mass., USA), cyano- gen bromide-activated Sepharose 4B and the Superdex 75 16/60 col- umn were from Pharmacia. Poly-L-proline was coupled to Sepharose beads at a concentration of 2 ~tmol/ml beads according to (Tanaka