A further study of the phospholipase-independent action of beta-bungarotoxin at frog end-plates.

1. The effect of beta-bungarotoxin (beta-BuTx) at the frog neuromuscular junction has been investigated further in order to distinguish more clearly between phospholipase- independent and phospholipase-dependent actions on transmitter release. 2. Inhibition of the enzymatic activity, by substitution of strontium for calcium, allowed determination of the dose-response curve of the early rapid decrease in transmitter release caused by the toxin. In the presence of strontium ions there was, however, still about 7% residual enzymatic activity, and electrophysiological evidence of it could be seen in room-temperature experiments at high concentrations of beta-BuTx. This residual enzymatic activity could be suppressed by lowering the temperature to 5 degrees C. 3. In normal calcium-Ringer solution beta-BuTx produced the typical triphasic effect on the amplitude of end-plate potentials (e.p.p.s). Lowering the temperature markedly delayed an then diminished the secondary transient increase. There was, however, comparatively little temperature influence on the first rapid decrease in e.p.p. amplitude. Enzymatic assays confirmed the temperature dependence of the toxin's phospholipase activity on model phospholipid substrates. 4. The kinetics of the phospholipase-independent action of beta-BuTx were examined in strontium-Ringer compared to calcium-Ringer solution, as well as in calcium-Ringer at different temperatures. Both the time to onset of inhibition and the time to 50% inhibition of the e.p.p., during the first phase of toxin action, are temperature-dependent and briefer in calcium than in strontium-Ringer solution. It is suggested that calcium is more effective than strontium in promoting this phospholipase- independent interaction of beta-BuTx with the nerve terminal membrane.