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ADP-Ribosylation with Clostridium Perfringens Iota Toxin

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ADP-Ribosylation with Clostridium Perfringens Iota Toxin Biochem. J. (1990) 266, 335-339 (Printed in Great Britain) 335 Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin Udo GEIPEL, Ingo JUST and Klaus AKTORIES* Rudolf-Buchheim-Institut fur Pharmakologie der Universitat GieBen, Frankfurterstr. 107, D-6300 GieBen, Federal Republic of Germany Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D- stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 /iM), at low concentrations of Mg2" (50 ItM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and -that the inhibitory effect is not due to inhibition of the polymerization of actin. INTRODUCTION perfringens type E strain CN5063, which was kindly donated by Dr. S. Thorley (Wellcome Biotech, Various bacterial ADP-ribosylating toxins modify Beckenham, Kent, U.K.) essentially according to the pro- regulatory GTP-binding proteins, thereby affecting cedure described (Stiles & Wilkens, 1986). Cytochalasin eukaryotic cell function. Pertussis toxin and cholera D was obtained from Sigma (Deisenhofen, Germany). toxin ADP-ribosylate GTP-binding proteins involved in DNAase I was a gift from Dr. H. G. Mannherz transmembrane signal transduction (for a review, see (Marburg, Germany). [oc-32P]ATP, [y-32P]ATP and Pfeuffer & Helmreich, 1988). Diphtheria toxin and [32P]NAD were prepared according to Walseth & Pseudomonas exotoxin A modify elongation factor II Johnson (1979) and Cassel & Pfeuffer (1978) respectively, (Collier, 1967; Iglewski & Kabat, 1975). In all of these or were obtained from NEN (Dreieich, Germany). All cases, the eukaryotic protein substrates of these toxins other reagents were of analytical grade and were pur- possess inherent GTPase activities, which are apparently chased from commercial sources. crucial for the regulatory functions of the GTP-binding proteins. Clostridium perfringens iota toxin belongs to a Purification of skeletal muscle actin novel class of ADP-ribosylating toxins which modify Rabbit skeletal muscle actin was purified as described actin (Simpson et al., 1987; Schering et al., 1988). ADP- & ribosylation inhibits the ability of actin to polymerize (Spudich Watt, 1971). (Schering et al., 1988). Additionally, ADP-ribosylated ADP-ribosylation of actin actin acts like a capping protein to inhibit polymerization Skeletal muscle actin was ADP-ribosylated as de- of non-modified actin at the fast-growing end of actin scribed (Aktories et al., 1986a) in a medium containing filaments (Wegner & Aktories, 1988). Actin is an ATP- 40 gM-CaCl2, 40 /tM-ATP, 1 mM-dithiothreitol, 50 /LM- binding protein and possesses inherent ATPase activity NAD 10 mM-triethanolamine/HCl, pH 7.5, and the in- (for a review, see Pollard & Cooper, 1986). It appears dicated concentrations of iota toxin (ADP-ribosylating that ATP hydrolysis plays an important role in the component) in a total volume of 100 gul for 1 h. dynamic transition between monomeric and polymeric actin. Polymerization of actin largely increases ATP Filter assay for determination of actin ADP-ribosylation hydrolysis. Since ADP-ribosylation of actin inhibits This was performed as described (Schering et al., 1988). polymerization, it is important to clarify whether ADP- ribosylation affects the ATPase activity of actin directly ATPase activity or indirectly. Basal ATPase activity of non-polymerized ATPase activity was determined essentially as de- G-actin is very low. The mycotoxin cytochalasin is scribed for GTPase activity (Aktories et al., 1982). After known to stimulate G-actin ATPase activity severalfold ADP-ribosylation of actin (in a volume of 100 ltl), free (Brenner & Korn, 1980; for a review, see Cooper, 1987); nucleotides were removed by the addition of 20,l of we therefore studied the effects of ADP-ribosylation on Dowex 1 x 8 (50 %). The samples were vortex-mixed and cytochalasin-stimulated actin ATPase activity. centrifuged for 1 min. The supernatant was removed. MgC12 (50 /M or 1 mM), cytochalasin D (1 /tM) and MATERIALS AND METHODS [y-32P]ATP (50 /tM, about 200000 c.p.m., all final con- centrations) were added to the supernatant. ATP hydro- Materials lysis was terminated after incubation for 2 h or as The ADP-ribosylating component of C. perfringens indicated at 37 °C by the addition of 450,l of ice-cold iota toxin was purified from the culture medium of C. sodium phosphate buffer (20 mm, pH 2) containing 5 00 * To whom correspondence should be addressed. Vol. 266 336 U. Geipel, I. Just and K. Aktories (w/v) activated charcoal to 50 ,l aliquots of the reaction 400 I iIIi mixture. After centrifugation for 15 min at 15000 g, - 300 ,ul of the supernatant was counted for radioactivity. 0 Binding of cytochalasin D to actin -o Actin (15 /SM) was incubated in G-buffer [2 mM-Tris/ E 300 HCl (pH 8.0)/0.2 mM-CaCl2/0.005 % NaN3/0.2 mm- ATP/0.5 mM-,8-mercaptoethanol] containing 110IM- Co.E NAD without or with iota toxin (3 /sg) for 45 min in a 0- 0 total volume of 180 Thereafter, MgCl2 (50 ftM) and 200 #1. I- cytochalasin D (1 /tM) were added to give a final volume 0 of 200 ,ll. The mixture was incubated for 1 h at room temperature. Then the mixture was centrifuged in a E 0 Centricon filter membrane holder (Amicon, Witten, 100 Germany) for 10 min at 4000 g. Thereafter, an aliquot of .2 the filtrate (5 1I) was used in the ATPase assay. 0C) : ~/ 0~ I Polyacrylamide-gel electrophoresis I- o ~zI-o-o-o-OQ This was performed under non-denaturing conditions 0 1 10 100 1000 according to Laemmli (1970), with the exception that the 11 % polyacrylamide gel contained no SDS but did [Cytochalasin D] (nM) contain 0.5 mM-ATP. Fig. 2. Inhibition of cytochalasin D-stimulated actin ATPase by Actin concentration ADP-ribosylation with iota toxin This was determined spectrophotometrically by Skeletal muscle actin was ADP-ribosylated in the presence using a specific absorption coefficient at 290 nm of of iota toxin (5,ug) and 50 ,M-NAD (0) or was not 0.62 ml- mg cm-'. ADP-ribosylated (0). Thereafter, the actin concentration was made up to 0.1 #M. Actin ATPase was stimulated by the indicated concentrations of cytochalasin D in the RESULTS presence of 1 mM-MgC12 and 50 tuM-[y-32P]ATP. Fig. 1 illustrates the influence of increasing concentrations of iota toxin on the ADP-ribosylation of 6.0,g of iota toxin/ml respectively. Fig. 2 shows that actin and on ATP hydrolysis stimulated by cytochalasin cytochalasin D increased ATP hydrolysis by skeletal D (1 /tM). ATP hydrolysis was determined in the presence muscle actin in a concentration-dependent manner. A of 50 4uM-MgCl2, a concentration which does not induce maximal 30-fold increase in ATP hydrolysis occurred at polymerization. ADP-ribosylation of actin inhibited 1 /LM-cytochalasin D. ADP-ribosylation of actin by iota actin-catalysed ATP hydrolysis in a concentration-de- toxin almost completely inhibited the cytochalasin- pendent manner. Half-maximal and maximal inhibition stimulated ATPase activity. In order to exclude further of ATPase activity was obtained with 0.4 ,ug and 6.0,ug the possibility that inhibition of ATPase activity was ofiota toxin/ml respectively. Half-maximal and maximal caused by ADP-ribosylation-induced inhibition of actin ADP-ribosylation of actin was observed at 1 ,ug and polymerization, cytochalasin-stimulated ATPase activity was studied at actin concentrations (0.1 /M) below its critical concentration. The MgCl2 concn. was 1 mm, a til 11 .. concentration which causes an increase in G-actin C ATPase activity. Fig. 3(a) illustrates the time course of 50 - O °-O- 0.8 N ATP hydrolysis by actin. Actin ATPase activity was 01) 0 constant for at least 150 min. In the absence of iota I.. 40 0 6_ toxin, NAD did not affect ATP hydrolysis; subsequent L2 addition of iota toxin almost completely inhibited ATP Co Hn 30 -0.4 m c 4- O hydrolysis. The time course of the ADP-ribosylation of 200 f actin shows that 2 min after addition of iota toxin, about 50%0 of the actin was ADP-ribosylated (Fig. 3b). The < E E 10 0 high rate of ADP-ribosylation correlates with the almost U-. immediate inhibition of ATPase. Inhibition of actin 0 0.02 0.2 2 20 E ATPase activity by ADP-ribosylation was revealed by [Iota toxin] (pg/ml) direct determination of the nucleotide bound to actin. For this purpose [a-32P]ATP-loaded actin was treated by Fig. 1. Influence of increasing concentrations of iota toxin on Dowex to remove unbound nucleotides. Then, actin was ADP-ribosylation of actin (0) and inhibition of actin ADP-ribosylated by iota toxin in the presence of cyto- ATPase (0) chalasin D for a further 60 min and the nucleotides Skeletal muscle actin (7/TM) was incubated for 2 h with the bound were determined by t.l.c. Fig. 4 shows that indicated concentrations of iota toxin. Thereafter, the unmodified actin bound almost only ADP. In contrast, ATPase activity of actin (5,M) was determined in the the ADP-ribosylated actin bound significantly more presence of I /uM-cytochalasin D, 50 /,M-[y-32P]ATP and ATP. Inhibition of stimulation of actin ATPase could be 50 /tM-MgCl2. due to the inability of cytochalasin to interact with ADP- 1990 ADP-ribosylation of actin by iota toxin 337 (a) c 0 .U o E <0 o- 0 ADP .0- C--a (b) I._ 1.0 c E 0.8 a) -5 0.6 ._ ADP ATP cL a- (0.4 a: a o '0.2 of actin z Fig.
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