Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes.

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. The resistance (greater than 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture of in suspension or whether choleragen was found at 0 or 37 degrees C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside GM1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [3H]GM1, up to 70% of the cell-associated GM1 was extracted under the mild conditions. When the Gm1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of GM1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [3H] galactose by immunoadsorption, only labeled GM1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.