Cholera toxin and the adenylate cyclase-activating signal.
Studies with chemically modified cholera toxin derivatives showed that all treatments that decreased the ability of toxin to bind to mouse thymus cells or to polystyrene-coupled GM1 ganglioside caused a concomitant reduction in the toxin's ability to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) in thymus cells and skin vascular permeability in rabbits. Dissociation of the H (heavy) and L (light) subunits abolished the biologic activity without inhibiting receptor binding, as did treatment with arginyl-specific reagents (which did not change the aggregation state of the toxin). When thymus cells were incubated with 125I-labelled toxin at 37C,only about 1% of the total cell-bound radioactivity was recovered in the cytosol supernate. Similar values were found for cells incubated with toxin at 0C, and with 125I-labelled choleragenoid at 37C or 0C. Thymus cells rapidly bound less than or equal to equal to 5 X 10(4) cholera toxin molecules per cell at both 0C and 37C. Much less, however, of the radioactive toxin bound at 37C than of that bound at 0C was displaced by addition of unlabelled toxin or choleragenoid. Similar temperature-related irreversible binding was noted with 125I-labelled choleragenoid. The relative amounts of H and L subunits in the irreversibly cell-bound and in the displaced 125I-labelled toxin were indistinguishable. Treatment of thymus cells at 37C, but not at 0C, with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide caused a 10-fold reduction of adenylate cyclase stimulation by cholera toxin without inhibiting activation by epinephrine or prostaglandin E1, or appreciably altering the basal, unstimulated enzyme activity. The carbodiimide inhibited the cyclic AMP response to cholera toxin when added shortly after the toxin had bound to the cells (early in the lag phase).
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004768:Enterotoxins / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects*; D004837:Epinephrine / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D051379:Mice; D012867:Skin / Q000187:drug effects; D013696:Temperature; D013950:Thymus Gland / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D014734:Vibrio cholerae
D001639:Bicarbonates; D004768:Enterotoxins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D005022:Ethyldimethylaminopropyl Carbodiimide; D004837:Epinephrine