EFFECT OF ß-BUNGAROTOXIN ON ATPase ACTIVITY, 45Ca++ UPTAKE AND CONFORMATION OF BIOLOGICAL MEMBRANES

Abstract

Effects of β-bungarotoxin (β-BuTX) on Ca++-activated ATPase, 45ca++ uptake and the conformation of biological membranes were studied in order to shed light on the mechanism of action of β-BuTX. β-BuTX possessed one sixth of the phospholipase A activity of the phospholipase A (PLA) of Naja naja atra venom. Thus, effects of PLA on Ca++-activated ATPase, 45ca++ uptake and the conformation of biological membranes were also studied for comparison. Both β-BuTX and PLA inhibited Ca++-activated ATPase of sarcoplasmic reticulum and microsomes of the rabbit skeletal muscle. Egg lecithin enhanced the inhibitory action of β-BuTX but antagonized that of PLA. Both ATP and K+ overcame partially but N-ethylmaleimide and reduced glutathione did not affect the inhibitory action of β-BuTX and PLA. β-BuTX markedly inhibited 45Ca++ uptake of sarcoplasmic reticulum and microsomes of the rabbit skeletal muscle. Brain synaptosome of the guinea pig was less susceptible than the sarcoplasmic reticulum and microsomes to the inhibitory action of β-BuTX. PLA was more potent than β-BuTX in inhibiting 45ca++ uptake. Effect of β-BuTX and PLA on membrane conformation was studied by the fluorescence probe method. β-BuTX changed the conformation of microsomes and mitochondria of the skeletal muscle by increasing 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. CaCl2 markedly enhanced this effect of β-BuTX on mitochondria, β-BuTX increased ANS fluorescence with synaptosomes only in the presence of CaCl2. ANS fluorescence with sarcoplasmic reticulum was not affected by β-BuTX even in the presence of CaCl2. By contrast, PLA decreased ANS fluorescence with sarcoplasmic reticulum and synaptosomes but did not affect that with microsomes and mitochondria. All of these findings suggest that β-BuTX may inhibit the release of acetylcholine from the motor nerve terminals by altering the membrane conformation associated with Ca++ transport. PLA either did not affect or affected differently the membrane conformation, so that it did not mimic β-BuTX action. β-Bungarotoxin (β-BuTX), a presynaptic neurotoxin from the venom of Bungarus multicinctus, blocks neuromuscular transmission by inhibiting acetylcholine release from the motor nerve terminal (Chang et al., 1973). The mechanism of the inhibitory action on acetylcholine release by β-BuTX is not known. It has been demonstrated that β-BuTX is potent in inhibiting 45ca++ uptake of the rat brain mitochondria (Wagner et al., 1974). It has been suggested that β-BuTX may inhibit acetylcholine release by a direct or indirect inhibition of mitochondrial 45ca++ uptake. The purpose of this investigation is to study the effects of β-BuTX on Ca++–activated ATPase activity, 45ca++ uptake and the conformation of biological membranes in order to explore the mechanism of action of β-BuTX. Effects of phospholipase A (PLA) on these biochemical activities were also studied for comparison.