Mechanism of action of cholera toxin on intact cells. Generation of A1 peptide and activation of adenylate cyclase.
When intact mouse neuroblastoma NB cells were incubated with choleragen at 4 degrees C, washed, and incubated at 37 degrees C, activation of adenylate cyclase occurred rapidly after a delay of 15 min. The cells were incubated under the same conditions with 125I-labeled toxin, lysed, and solubilized with sodium dodecyl sulfate under mild conditions. Soluble proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of dithiol reductants to separate labeled toxin products. Initially, only 0.1 to 0.2% of the cell-associated radioactivity migrated on the gels as the A1 peptide of choleragen. After a 15-min delay, the amount of A1 peptide increased rapidly with time and paralleled the activation of adenylate cyclase. Similar results were observed with human skin fibroblasts, Friend erythroleukemic cells, and II3-alpha-N-acetylneuraminosyl-gangliotetraosylceramide-treated rat glioma C6 cells. When toxin-treated NB cells were incubated at increasing temperatures, generation of A1 peptide and activation of adenylate cyclase increased in parallel. Both processes were prevented by incubation of cells at 4 or at 37 degrees C in the presence of anticholeragen antibodies. These results indicate that there is delay both in the formation of A1 peptide and in the activation of adenylase cyclase in intact cells. As A1 is believed to be the catalytically active component of choleragen, it is suggested that the lag period may be related in part to the time required to generate A1 peptide from choleragen.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D010446:Peptide Fragments; D002772:Cholera Toxin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide