Bacterial toxins as probes for membrane glycolipids in mammalian cells.

Cholera toxin which binds specifically and with high affinity to a glycolipid; ganglioside GM1, has been used as a probe to study glycolipid-protein interactions in the plasma membranes of BALB/c 3T3 mouse fibroblasts and mouse lymphocytes. Evidence is presented that: i) a proportion of cholera toxin bound to GM1 of BALB/c 3T3 cells withstood extraction with Triton X-100 at 0C and remained associated with the cytoskeleton; ii) at 37C, cholera toxin-GM1 complexes were completely extracted with Triton X-100. The resulting complexes existed in a macromolecular form that did not involve other membrane components; iii) antibody-induced capping of GM1-cholera toxin complexes in mouse lymphocytes had characteristics in common with cell surface immunoglobulin- and Con A-capping systems. Immuno- fluorescence studies revealed that in all three systems, capping was accompanied by the redistribution of cytoskeletal components and was inhibited by drugs that inhibit microfilament, but not microtubule, function. These findings suggest that capping in these systems occurs by contractile mechanisms with common features and further suggest that, under certain circumstances, some form of trans-membrane linkage exists between GM1 and the cytoplasm. The role of gangliosides as receptors for tetanus toxin in rat brain membranes was investigated Tris-acetate buffer, pH 6.0 and in a more "physiological" buffer. In agreement with the findings of previous workers, the binding of 125I-labelled tetanus toxin to rat brain membranes in Tris- acetate buffer, pH 6.0 was of high affinity and readily inhibited by gangliosides. The receptors were sensitive to sialidase but less so to heat or protease. In contrast, toxin binding to rat brain membranes in Krebs-Ringer buffer, pH 7.4 involved high and lower affinity components. These receptors were fewer, and markedly sensitive to proteases, heat and sialidase. Binding was not readily inhibited by ganglioside so protein, rather than ganglioside, is likely to represent the high affinity binding site detected in Krebs- Ringer buffer.