Multiple Roles of Erythrocyte Supernatant in the Activation of Adenylate Cyclase by Vibrio cholerae Toxin in Vitro

Dm, Gill

doi:10.1093/infdis/133.supplement_1.s55

Cited by 94 publications

(23 citation statements)

References 5 publications

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“…Thus, half-maximal activation of adenylate cyclase, when measured at 1.5, 24, and 48 hr, required 200, 1.1, and 0.35 pM choleragen, respectively. These results are consistent with a catalytic model of choleragen action as first suggested by Gill [12][13][14], in which a few toxin molecules bound to the cell progressively activate more and more adenylate cyclase. Such a mechanism has been demonstrated in disrupted cells with A~ peptide [12] but not previously in intact cells.…”

Section: Discussionsupporting

confidence: 91%

“…These results are consistent with a catalytic model of choleragen action as first suggested by Gill [12][13][14], in which a few toxin molecules bound to the cell progressively activate more and more adenylate cyclase. Such a mechanism has been demonstrated in disrupted cells with A~ peptide [12] but not previously in intact cells. In contrast, Cuatrecasas and coworkers proposed that the activation process involved a bimolecular interaction between the toxin and adenylate cyclase, each toxin molecule activating one cyclase [1,2,30].…”

Section: Discussionsupporting

confidence: 91%

“…The initial event in toxin action is its binding to specific receptors on the cell surface that have been identified as the ganglioside GM1115, 6,11,19,23,26,28]. In intact cells, there is a discrete lag period between choleragen binding and activation of adenylate cyclase [2,14,22] which is not observed in disrupted cells [12]. More recently, the mechanism of the activation process in disrupted cells has been eluc.idated.…”

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confidence: 99%

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Mechanism of action of cholera toxin: Studies on the lag period

Fishman

1980

J. Membrain Biol.

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The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside GM1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1, and 0.35 PM, respectively. Under the latter conditions, each cell was exposed to 84 molecules to toxin. The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 degrees C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In GM1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 degrees C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 degrees C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for less than 10 min at 37 degrees C accumulated cyclic AMP when shifted to 15 degrees C. Under optimum conditions at 37 degrees C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for choleragen action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 91%

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confidence: 99%

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Mechanism of action of cholera toxin: Studies on the lag period

Fishman

1980

J. Membrain Biol.

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“…In addition, both choleragen and LT require NAD for activation of adenylate cyclase in disrupted cells (9,11,12) and possess NAD glycohydrolase and ADPribosyltransferase activities (13)(14)(15). It has been proposed that both toxins exert their effects through the NAD-dependent ADP-ribosylation of either adenylate cyclase itself or of a protein critical to cyclase activation (9,11,(13)(14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Moss¹,

Garrison²,

Fishman³

et al. 1979

J. Clin. Invest.

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A B S T R A C T Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin. These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides. After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraininyl]-galactosylglucosylceramide (GM,), the cells responded to E. coli heat-labile enterotoxin. The di-and trisialogangliosides were considerably less effective. GMI, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E. coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin. This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin.

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“…The effects of the toxin are a result of the intracellular accumulation of cyclic AMP and activation of adenylate cyclase (6-13); Gill et al (13), with a cell-free system derived from pigeon erythrocytes, have shown that the toxin activates adenylate cyclase in a process that is dependent upon NAD and ATP. Thus, the biochemical effects of the toxin are similar to those of choleragen (cholera toxin), an enterotoxin of Vibrio cholerae, which also causes the NADdependent activation of adenylate cyclase (14,15). Although the mechanism of choleragen activation and NAD utilization has not been determined, it has been demonstrated (16)(17)(18) that both the holotoxin and its A protomer can catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide (reaction [1]) and the transfer of the ADP-ribose moiety of NAD to arginine (reaction [2]).…”

Section: Introductionmentioning

confidence: 99%

Activation of Adenylate Cyclase by Heat-Labile Escherichia Coli Enterotoxin

Moss¹,

Richardson²

1978

J. Clin. Invest.

157

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A B S T R A C T Highly purified, polymyxin-released, low molecular weight Escherichia coli heat-labile enterotoxin (LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity was stimulated by dithiothreitol and was independent of cellular components. Nicotinamide formation was enhanced by arginine methyl ester > D-arginine -L-arginine -guanidine. A 20-fold increase in activity was noted with arginine methyl ester, and maximal activity again required dithiothreitol. When the reaction was initiated with toxin, a delay was observed before a constant rate was established. The reaction products found after incubation of [adenine-U-14C]NAD and L-[3H]arginine or unlabeled arginine methyl ester with the enterotoxin had mobilities on thin-layer chromatograms similar to the reaction products obtained after incubation of choleragen with these substrates and are consistent with the formation of ADP-ribose-L-arginine and ADP-ribose-L-arginine methyl ester, respectively. Both toxins, which catalyze the NAD-dependent activation of adenylate cyclase, thus appear to possess NAD glycohydrolase and ADP-ribosyltransferase activities. Although the activities of both toxins are dependent on dithiothreitol, Escherichia coli enterotoxin exhibited optimal activity in Tris(Cl-) (pH 7.5) and was inhibited by high concentrations of potassium phosphate (pH 7.0) or low pH (sodium acetate, pH 6.2). It appears Portions of this work were presented at the meetings of

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Multiple Roles of Erythrocyte Supernatant in the Activation of Adenylate Cyclase by Vibrio cholerae Toxin in Vitro

Cited by 94 publications

References 5 publications

Mechanism of action of cholera toxin: Studies on the lag period

Mechanism of action of cholera toxin: Studies on the lag period

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Activation of Adenylate Cyclase by Heat-Labile Escherichia Coli Enterotoxin

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