The binding of two classes of neurotoxins to axolemma of mammalian brain.

The binding of a 3H-labeled ethylenediamine derivative of tetrodotoxin ([3H]EN-TTX) and 125I-labeled polypeptide neurotoxins, purified from the sea anemone Anenomia sulcata (ATXII) and the scorpion Androctonus australis Hector (AaHII), was studied using axolemma-enriched membrane fractions. The membrane fractions were derived from a purified preparation of myelinated axons fractionated via a linear sucrose gradient in a zonal rotor. The specific activity of Na+K+ ATPase in the axolemma-enriched preparation, found in the 28-32% sucrose region of the density gradient, was 59.1 mumol of ATP hydrolyzed/mg of protein/h. As estimated by 3H-specific ouabain binding, this fraction contained 183.6 pmol of Na+K+ ATPase/mg of protein. The 28-32% region of the density gradient was most enriched in the binding capacity for all neurotoxins, while the stoichiometry of the binding activities varied throughout the density gradient. The maximal binding (Bmax) of [3H]EN-TTX was 1 pmol/mg; the dissociation constant (KD) of the neurotoxin for its receptor was 2 X 10(-10) M. The comparable values for ATXII were 3.2 pmol/mg and 1.5 X 10(-7) M, respectively, while AaHII had a Bmax of 0.08 pmol/mg and a KD of 3.3 X 10(-9) M. The relationship of the binding of these neurotoxins to that observed in other axonal plasma membrane preparations is discussed.