Neuropharmacological characterization of fragment B from tetanus toxin.
Tetanus toxin and Fragment B from tetanus toxin were assayed for activity on the mouse phrenic nerve-hemidiaphragm preparation. Both molecules produced blockade of neuromuscular transmission, but the parent molecule was at least two orders of magnitude more potent than the fragment. Experiments were done to determine whether the toxicity attributed to Fragment B was authentic or due to contamination with the parent molecule. Analysis of the fragment by high-performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed trace contamination. Removal of the major contaminant (1-2%) did not abolish toxicity of the material. However, in pharmacological experiments with native toxin and its fragment, the latter behaved indistinguishably from the former. At equiactive concentrations, both were antagonized by Fragment C and both were antagonized by lysosomotropic agents (ammonium chloride and methylamine hydrochloride). In addition, monoclonal antibodies directed against epitopes in Fragment C neutralized both native toxin and the material presumed to be Fragment B. The studies with antagonists and antibodies suggest that the toxicity apparently associated with Fragment B was in fact due to trace contamination with the parent molecule. In experiments on planar lipid bilayers, Fragment B formed pH-dependent channels. This activity was not abolished by monoclonal antibodies directed against epitopes in Fragment C. The data indicate that Fragment B retains the ability to form channels in membranes, but in the absence of Fragment C it retains little ability to paralyze neuromuscular transmission.
D000818:Animals; D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D051379:Mice; D009469:Neuromuscular Junction / Q000187:drug effects; D010446:Peptide Fragments / Q000032:analysis*; D051381:Rats; D013329:Structure-Activity Relationship; D013744:Tetanus Toxin / Q000032:analysis*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D013744:Tetanus Toxin