Calcium ion independent membrane leakage induced by phospholipase-like myotoxins

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.