Pharmacological experiments on the binding and internalization of the 50,000 dalton carboxyterminus of tetanus toxin at the cholinergic neuromuscular junction.

Experiments were done to compare the binding and subsequent internalization of the intact tetanus toxin molecule and of the isolated binding component from the molecule (i.e., 50,000 dalton carboxyterminus). In all respects, the isolated component behaved like the intact molecule. Both the holotoxin and the isolated carboxyterminus bind to neuromuscular preparations in a way that is only poorly dissociable. At low temperature (4 degrees C), the holotoxin remains at or near the cell surface and thus remains accessible to the neutralizing effects of polyclonal antibody. At the same temperature, the isolated binding component remains associated with receptors and thus antagonizes binding of the intact toxin. Warming tissues (35 degrees C) slowly promotes internalization of the toxin and of the isolated binding component, and nerve stimulation more rapidly promotes internalization. Ammonium chloride and methylamine hydrochloride antagonize the process by which nerve stimulation promotes internalization of bound protein. Data from chase experiments suggest that the receptor for tetanus toxin either has a relatively rapid turnover rate or exists in relative excess. Data from monoclonal antibody experiments show that, even when the toxin is bound to nerve endings, antibodies directed against epitopes in the carboxyterminus can cause appreciable neutralization. The most important implication of the data is that the isolated carboxyterminus of tetanus toxin can be used as a pharmacological tool to target drugs of interest to nerve endings.