Complete purification of beta-bungarotoxin. Characterization of its action and that of tityustoxin on synaptosomal accumulation and release of acetylcholine

Abstract

beta-Bungarotoxin, a snake venom protein (molecular weight 21 000) that irreversibly blocks release of acetylcholine from nerve terminals, was purified to homogeneity by ion-exchange chromatography and isoelectric focussing. Sodium dodecyl sulphate gel electrophoresis under reducing conditions resolved two subunits of molecular weight 11 400 and 9000. In the presence of deoxycholate, it showed phospholipase activity which was activated by Ca2+ but not Sr2+.beta-Bungarotoxin and tityustoxin, a polypeptide that prolongs the opening of sodium channels, inhibited choline accumulation by synaptosomes purified from rat cortex. Both toxins also induced release of acetylcholine which was maximal in the presence of Ca2+ and showed ED50 values of 5 . 10(8) and 10(6) M, respectively. Unlike tityustoxin, beta-bungarotoxin also induced release of choline and cytoplasmic lactate dehydrogenase from synaptosomes, with similar potency, suggesting that it causes some membrane disruption, following its binding to the membrane. The effects of tityustoxin on both accumulation and release were antagonised by tetrodotoxin, which specifically blocks Na+ channels, indicating that it mediates these effects by depolarization. Thus, these toxins may prove to be useful probes for characterisation of nerve membrane components involved in triggering transmitter release.