Solubilization and partial characterization of the tetrodotoxin binding component from electric eel electroplaque

Abstract

The tetrodotoxin (TTX) binding component from the electroplaque of Electrophorus electricus was solubilized and studied using ^3H-TTX purified by a modified procedure using Bio-Rex-70 at pH 8.7. The best extraction yield was obtained from a solubilization in 1-2% Lubroi-PX, at pH 7.6, for 2 hours, at a membrane fragment concentration equivalent to 2.0 - 2.5 g original wet weight tissue/ml. Gel filtration on Sepharose-6B was used to verify that the TTX binding component was solubilized. The molecular weight of the solubilized component was found to be about 200,000 using 10-43% glycerol gradients. A Sephadex G-50 assay was used to study binding. A K_D of 1.2 nM was obtained for ^3H-TTX binding. This binding was affected by cations in a manner similar to that found by Reed and Raftery [Biochemistry 15:944-953 (1975)]. The solubilized material was found to be unstable with respect to TTX binding; this stability was affected by the presence of cations and TTX, but not veratridine or procaine.