MOLECULAR PHARMACOLOGY, 20:565-570 565 All rights of reproduction in any form reserved. Characterization of Fragment C and Tetanus Toxin Binding to Rat

The binding characteristics of ‘251-labeled tetanus toxin and ‘251-labeled fragment C to rat brain membranes have been studied. Fragment C is a proteolytically derived portion of the holotoxin. Tetanus toxin is slightly more potent than fragment C in displacing 1251 labeled tetanus toxin bound to rat brain membranes. Conversely, fragment C is more effective than toxin in displacing bound ‘25I-labeled fragment C. However, the binding curves for the two compounds are in both cases parallel, suggesting that they compete for the same site. The equilibrium affinity constants for the binding of tetanus toxin and fragment C as obtained by both direct and indirect binding experiments were found to be in the nanomolar range (2-12 nM). This relatively high affinity is in contrast with the large number ofbinding sites (estimated as 2-9 nmoles/mg ofprotein). This concentration is consistent with the reported content of the gangliosides, GT1 and GD1b, in rat brain. The specificity of the toxin binding site has been tested; a number of putative neurotransmit-ters as well as inhibitors of axonal transport were unable to affect the binding at concentrations several orders of magnitude higher than the toxin or fragment C. The anticonvulsant drug diazepam, reported useful in the treatment of human tetanus, was equally inactive in displacing ‘25I-labeled tetanus toxin binding. The possible pharmaco-logical relevance of the binding site for tetanus toxin and this fragment is discussed