Binding choleragen

Figure 4. Effect of butyrate treatment on choleragen binding to HeLa cells...

A Cells exposed to medium containing 5mM sodium butyrate for times indicated. B After 48 hr, as in A, medium replaced with fresh medium without butyrate. C Cells exposed for 48 hr to medium containing the indicated concentrations of butyrate. Specific binding of, 2sI-choleragen determined as described in Ref. 5. (Data from Ref. 5J... 

Figure 5. Effect of labeled and unlabeled choleragen concentrations on n5I-choleragen binding to control and butyrate-treated HeLa cells...

The induction of toxin receptors by butyrate also occurred in serum-free medium (5). Serum contains gangliosides including GM1 and GM1-deficient cells can absorb GM1 from serum and become responsive to choleragen (22). Cells exposed simultaneously to butyrate and cyc1oheximide""cTid not exhibit an increase in choleragen binding (unpublished observations). Thus, butyrate appears to induce toxin receptors de novo in a manner analogous to the induction of GM3. 

Table II. Binding of Choleragen to HeLa Cells Treated with Various Fatty Acids ...

Table III. Binding of Choleragen to Friend Erythroleukemic Cells Treated with Sodium Butyrate or Dimethylsulfoxidea...

If GM1 is the choleragen receptor, then butyrate-treated cells should have an increase in GM1 content. This is demonstrated in Table IV. Although GM1 could not be detected in control HeLa cells, they would contain less than 1 pmol per mg protein based on the limits of the sensitivity of the analytical procedure (5). GM1 was quantitated in the butyrate-treated cells (28.5 pmol per mg protein) and this increase is similar to the 32-fold increase in toxin binding observed in cells from the same experiment. The delipidated residue contained less than 1% of the toxin binding found in intact cells (Table IV). In addition, removal of the cells from the culture dishes with trypsin as opposed to the mechanical scraping routinely used had no effect on 125I-cholera-gen binding to either control or butyrate-treated cells (5). 

Table V. Effect of Gangliosides on Binding of Choleragen to Cells ...

The above results indicate that the increased choleragen binding to butyrate-treated HeLa cells is associated with increased GMl content. This was confirmed with Friend erythroleu-kemic cells (Table VII). Untreated Friend cells have measurable... 

We consider here some pathologies of the G-protein-dependent signal pathways. Let us first consider the mechanism of action of the cholera toxin, secreted by the intestinal bacterium Vibrio cholera. Cholera is an acute diarrheal disease that can be life threatening. It causes voluminous secretion of electrolytes and fluids from the intestines of infected persons. The cholera toxin, choleragen, is a protein composed of two functional units—a B subunit that binds to gangliosides of the intestinal epithelium and a catalytic A subunit that enters the cell. The A subunit... 

Activation of adenylate cyclase by choleragen results from the toxin-catalyzed ADP-ribosylation of a regulatory component of the cyclase system, Gsa, a guanine nucleotide-binding protein involved in stimulation of the cyclase catalytic unit (1). The ADP-ribosylation reaction and cyclase activation are enhanced by soluble and membrane components (2-9). One membrane protein, known as ADP-ribosylation factor or ARF, was extensively purified by Kahn and Gilman and shown to be a GTP-binding protein (8,9). 

Fig. 2. A guanine nucleotide-binding protein cascade participates in the activation of adenylate cyclase by choleragen.

In the absence of other proteins, choleragen binds specifically to liposomes containing ganglioside That choleragen induced changes in the perme-... 

Epidemic cholera is a common cause of illness in countries with inadequate water treatment facilities. Cholera excretes a toxin (choleragen) consisting of a binding agent that attaches the microbe to the intestinal wall. The microbe then penetrates mucus layers, which produces the classic symptom of diarrhea. In the case of Asiatic cholera, untreated cases of infection have an approximate fatality rate of over 60 percent. Diarrhea, vomiting, and malaise appear after an incubation period from 12 hours to 3 days, symptoms caused by severe water loss and subsequent lack of body electrolytes. 

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