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Title: The interaction of tetanus toxin with adrenal chromaffin cells
Author: Colville, Caroline Anne
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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Tetanus toxin exerts its primary biological effect by impairing the release of inhibitory neurotransmitters in the central nervous system; the mechanism of this blockade, however, remains unknown. Studies using adrenal medullary chromaffin cells, which are closely related to the toxin's target neurones but much more accessible to biochemical investigation, have the potential to provide information on various aspects of the intoxication process of tetanus. Nicotine-evoked (but not basal) secretion of catecholamines from intact chromaffin cells was inhibited by tetanus toxin in a dose-dependent fashion up to a maximum of 75%, half-maximal inhibition being achieved at 0.7 nM toxin (in single- or double-chain form). The time course of this inhibition was long, approximately 16 hours. Catecholamine release evoked by Ba2+ ions was not affected by the toxin, while its effect on K+ -evokedsecretion was never greater than 30%. Pre-incubation ofcells with ganglioside GT1, a specific ligand of the toxin, in the absence of toxin itself inhibited nicotine-evoked release of catecholamines (50&37 inhibition with 24 uM GT1), making it impossible to evaluate the role of GT1 in mediating the action of the toxin. Tetanus toxin (radiolabelled) was also shown to bind in a specific fashion to chromaffin cells. Toxin binding under less physiological conditions of pH and ionic strength was a higher capacity (Bmax 0.7-1.2 pmol/mg protein) than that found under more physiological conditions (Bmax 0.2-0.3 pmol/mg protein); this is also characteristic of toxin binding to synaptic membrances. In both cases there appeared to be at least two components to toxin binding, a higher affinity component with a Kd value of approximately 1nM (which did not account for more than 20% of total binding capacity), and a lower affinity binding with a Kd value of 10-25 nM. As expected, pre-incubationof chromaffin cells with ganglioside GT1 enhanced their toxin-binding capacity, but did not noticeably affect Kd value, perhaps indicating that gangliosides mediate binding of tetanus toxin to untreated chromaffin cells. This was further suggested by the finding that neuraminidase treatment of cells markedly reduced toxin-binding capacity (by 50% at pH 7.4, 90% at pH 6.0) and also by the observation that pre-incubation of 125I-labelled tetanus toxin with GT1 led to a reduction in its binding to chromaffin cells. Mild trypsinisation of chromaffin cells completely abolished toxin-binding under both sets of conditions, suggesting the involvement of a protein component in toxin binding also.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available