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In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (M, 42,000 The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.] by cholera toxin involves the interaction of some part of the cyclase system exposed on the inner surface of the plasma membrane with NAD, a nucleoside triphosphate, and a cytoplasmic protein (1-4) and is conveniently studied in lysed pigeon erythrocytes. The likelihood that the NAD might function as an adenosine diphosphoribose donor in a toxincatalyzed reaction of the type NAD+ + protein T ADP-ribosyl protein + nicotinamide + H+ was increased by the demonstrations that the active A1 fragment of cholera toxin catalyzes the slow hydrolysis of NAD to ADP-ribose and nicotinamide (5) and the transfer of ADP-ribose to arginine and related compounds (6) and to itself (C. King, personal communication; ref. 7).If the postulated acceptor were adenylate cyclase or an associated membrane protein, it should be possible to demonstrate a toxin-dependent transfer of ADP-ribose from radioactive NAD to a small number of sites on pigeon erythrocyte ghosts. There is, however, a large toxin-independent incorporation of ADP-ribose which was difficult to decrease without losing the toxin response. We now report the resolution of this problem and the demonstration of several toxin-specific ADP-ribose acceptors, principally a 42,000 Mr membrane protein. METHODSThe medium used throughout consisted of 0.13 M NaCl, 0.01% sodium azide, Trasylol (aprotinin: FBA Pharmaceuticals) at 2 kallikrein inactivator units/ml, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), and NaOH to give pH 7.3 at 370. Washed and purified pigeon erythrocytes suspended in an equal volume of this medium were lysed by rapid freezing and then thawing. The lysate was centrifuged at 10,000 X g for 5 min to separate ghosts and cytosol. The cytosol was mixed with well-washed pig brain NAD glycohydrolase (Sigma) at a ratio of 1 ml/10 mg, agitated at 370 for 30 min, and then centrifuged to remove the enzyme. The ghosts were washed three times in 20 vol of medium. There are about 8 X 109 ghosts per ml (packed).Portions of lysate reconstituted from 1 part washed ghosts and 2 parts NAD-depleted cytosol fraction were incubated for 30 min at 250 or 370 with 10 mM thymidine, 5

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP‐ribosylates a eukaryotic GTP‐binding protein of the ras superfamily. We show now that the bacterially‐expressed product of the human rhoC gene is ADP‐ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP‐ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p2l1.ADP-ribosylating toxins have proved to be valuable tools for studying their target proteins. The targets generally suffer important changes in function upon modification, and they become labeled when radioactive NAD is used. Most of the known ADP-ribosylating toxins have GTP-binding proteins (G proteins [16]) as substrates and, although the reason is not clear, this association leads to the speculation that undiscovered toxin-G protein pairs may exist. Diphtheria toxin and exotoxin A of Pseudomonas aeruginosa modify elongation factor 2, a 94-kilodalton (kDa) GTP-binding protein, and cholera and pertussis toxins ADP-ribosylate G proteins of about 40 kDa (GS, Gi, G., and transducin). However, no bacterial enzyme has been found that efficiently modifies G proteins of the 21-kDa size class such as the products of genes H-ras, K-ras, and N-ras (13), R-ras (20), rho (21), and ral (6), the proteins YPT1 (29) and Gp (9), or the activator of cholera toxin known as ARF (19) and S (14).In this report, we describe the substrate and some physiological effects of a clostridial enzyme that specifically transfers ADP-ribose to a GTP-binding protein (p21.bot) common to a wide variety of cells and tissues. While the work was in progress the enzyme was described by Aktories et al. (2), who designated it C3. We use the same name. It has several attributes of an enzymatically active portion of a toxin, but since no toxic activity has been demonstrated, we describe it provisionally as an exoenzyme. We show that the reaction catalyzed is indeed an ADP-ribosylation, that the material identified as C3 is in fact responsible, and that ADP-ribosylation of intracellular p21.bot has functional consequences.C3 is secreted only by the C and D types of Clostridium botulinum. These two types also differ from the more commonly studied A, B, and E types in other respects. In particular, the structural information for their Cl and D neurotoxins is carried by lysogenic or pseudolysogenic bacteriophages and nontoxinogenic cured strains are relatively easy to generate (8). Most C and D strains also elaborate C2 toxin that is enterotoxic, cytopathic, and lethal but not neurotoxic. Its enzymatic subunit catalyzes the ADP-ribosy-* Corresponding au...

By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. diffcile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin. Clostridium difficile induces its pathogenic effects by