High affinity binding of tetanus toxin to mammalian brain membranes.

A sensitive binding assay was developed to study tetanus toxin binding to brain membranes in vitro. Binding of '2"I-tetanus toxin was linear with tissue content only at very dilute tissue concentrations, 0-20 ng membrane protein/200 pl for bovine cortex membranes. At these low tissue concentrations '261-tetanus toxin displayed high affinity, single phase binding with a dissociation constant (KD) of 1.2 n~ and no apparent cooperative effects. The possibility that a protein was involved in the high affinity binding of tetanus toxin to brain membranes was explored by the use of proteolytic enzymes and amino acid specific reagents. Treatment of cortex membranes with enzymes such as achymotrypsin and trypsin as well as protein modification reagents such as iodoacetamide, ethoxyformic anhydride, N-ethylmaleimide, and N-bromosuccinimide did not alter '251-tetanus toxin binding to bovine cortex membranes, suggesting that proteins do not play a major role in the tetanus toxin receptor. Gangliosides competed for toxin binding, some with nanomolar potency such as N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyll-galactosylglucosylceramide ( h b ) (Ki = 6 m) and galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GD,~) (ICi = 10 m). Other gangliosides such as galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Ki = 450 m) and Nacetylgalactosaminyl[ N acetylneuraminyll-galactosylglucosylceramide (Xi > 10,000 m) were less active. These data suggest that gangliosides with structures similar to Gnb or G ~ l b might function as the physiologic receptor site. lZ51-Tetanus toxin binding displayed a heterogeneous regional distribution in bovine brain with highest levels in areas rich in gray matter such as hypothalamus, cortex, and cerebellum. Areas high in white matter such as medulla, corpus callosum, and optic tract showed about ' /e as much binding. The receptors for tetanus toxin appear concentrated at the synapse since synaptic plasma membranes bound about 10-fold more toxin than axonal membranes prepared from bovine brain.