Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom
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B. Lomonte, A. Tarkowski and L. Å. Hanson. Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom. Toxicon 32, 1359-1369, 1994.-The cytotoxic activity of Bothrops asper myotoxin II, a lysine-49 phospholipase A2 isoform, on different cell types in culture, was investigated. Myotoxin II caused a dose-dependent cytolytic effect on all cell types tested, characterized by rapid release of cytoplasmic lactic dehydrogenase and drastic morphological cell alterations. Quantitative differences in the susceptibility to myotoxin II among cell types fell within a relatively narrow range, and in general, the toxin was cytolytic at concentrations of 50-100 μg/ml (3-7 μM), when assays were performed using culture medium as a diluent. Toxin activity was markedly enhanced if phosphate-buffered saline was utilized instead of medium. The cytotoxic activity of myotoxin III, an aspartate-49 isoform from the same venom, on both endothelial cells and skeletal muscle myoblasts was higher than that of myotoxin II, suggesting that, although phospholipase A2 activity is clearly not required for the induction of cell damage, it may have an enhancing role. In contrast to B. asper myotoxins, other basic phospholipases A2 with myotoxic activity in vivo (notexin from Notechis scutatus, and two enzymes isolated from Vipera russelli venom) did not affect endothelial cells and myoblasts. Pretreatment of cells with neuraminidase, tunicamycin, or protamine, did not alter their susceptibility to myotoxin II. At low temperatures (2-4°C) myotoxin II was devoid of cytolytic effect. Washing and neutralization experiments using heparin with low affinity for antithrombin or mouse monoclonal antibody MAb-3 suggest that at low temperatures myotoxin II binds very weakly to the cells, and that its normal interaction with the putative target is probably not only based on charge, but that a membrane penetration event may be required