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Title: The interaction of GM1 ganglioside with cholera toxin on membranes of cells
Author: Fletcher, Marianne Elizabeth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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Cholera toxin (molecular weight 84kD) binds with high affinity (Kd = 10 -9M) to GM1 ganglioside on the outer surface of most eukaryotic cells before all or part of the molecule is internalised and activation of adenylate cyclase occurs. The GM1 ganglioside is believed to diffuse laterally on the cell surface. There is also evidence to suggest that cholera toxin requires multivalent binding to GM1 before it can activate adenylate cyclase. The effect of cholera toxin binding on the lateral diffusion of GM1 was examined using the Fluorescence Recovery after Photobleaching technique either with fluorescently labelled toxin or with inserted, fluorescently labelled GM1 ganglioside. Both toxin-receptor complex and receptor alone showed the same percentage mobility (about 60-70%) on the surface of the NIH 3T3 cells (a fibroblast cell line) and both had a lateral diffusion coefficient of about 1 x 10-9 cm2s-1. This result shows that bound toxin mobility does not differ from inserted ganglioside mobility. An interpretation of the results may be that GM1 molecules were compartmentalised on the fibroblast cell surface into mobile and immobile areas. The involvement of non-coated invaginations in cholera toxin internalisation was confirmed by preliminary binding experiments with colloidal gold conjugated cholera toxin. The cholera toxin was also used as a probe to locate GM1 intracellularly by the Post-Embedding Immunogold technique on mouse small intestine (target tissue for cholera toxin). A previously unreported, specific binding to the heterochromatin of the nucleus of mouse intestinal cell was discovered. The intracellular localisation of GM1 has previously mainly been studied by cell fractionation studies which indicated that only a small amount of total cell ganglioside is found within the nucleus. This binding of cholera toxin to the nucleus was further investigated using biochemical binding studies which also appeared to indicate a specific binding site for the toxin within the nucleus which has not been fully characterised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available