Molecular Pharmacology, Vol 20, 565-570, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Characterization of Fragment C and Tetanus Toxin Binding to Rat Brain Membranes

¹ Bacterial Toxins Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, and Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20205

The binding characteristics of ¹²⁵I-labeled tetanus toxin and ¹²⁵I-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 ¹²⁵I-labeled tetanus toxin bound to rat brain membranes. Conversely, fragment C is more effective than toxin in displacing bound ¹²⁵I-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 of binding sites (estimated as 2-9 nmoles/mg of protein). This concentration is consistent with the reported content of the gangliosides, G_T1 and G_D1b, in rat brain. The specificity of the toxin binding site has been tested; a number of putative neurotransmitters 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 ¹²⁵I-labeled tetanus toxin binding. The possible pharmacological relevance of the binding site for tetanus toxin and this fragment is discussed.