Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS–PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A 2. B. lanceolatus venom (50 μg/ml) and the caseinolytic protein (20 μg/ml) produced contracture and progressive irreversible blockade (50% in 25±5 min (SEM) and 45±15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 μM, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4 mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B. lanceolatus venom and the purified protein caused depolarization which was prevented by d-tubocurarine (14.6 mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.