Effects on cultured mammalian cells of myotoxin III, a phospholipase A2 isolated from Bothrops asper (terciopelo) venom
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Myotoxin III (MT-III), a myotoxic phospholipase A2 from Bothrops asper, was studied with respect to interactions with cultured mammalian cells and red blood cells. Tests of the cytopathogenic effect of MT-III on different cell lines indicated that rat skeletal muscle L6 myoblasts were more sensitive to the toxin than chinese hamster ovary cells, human lung fibroblasts, mouse adrenal tumour cells and rat intestinal epithelial cells. Specific plasma-membrane permeabilization was assayed as release of a cytosolic [3H]uridine nucleotide marker from toxin-treated L6 cells. A dose- and time-related membrane permeabilization was induced at 37°C, but not at 0°C. A half-maximal effect was obtained after 20 min. 30 μg/ml MT-III induced 50% marker release in 1 h, and the effect was not reversed by post-incubation for up to 48 h in toxin-free medium. The membrane permeabilization in L6 cells did not seem to require cellular internalisation of the toxin. The catalytic site of the toxin was inactivated by alkylation with p-bromophenacyl bromide (BPB). This treatment abolished the toxin's specific PLA2 activity, as assayed in vitro, and reduced the PLA2 activity on the myoblast membrane by more than 95%, as measured by release of [14C]arachidonic acid from prelabelled cells. However, the membrane-permeabilizing effect (release of cytosolic marker) was reduced only by 70% upon modification with BPB. We also report that MT-III is not directly haemolytic, and one reason for this is the inability of the toxin to associate with the membranes of human or mouse erythrocytes. Taken together, the data suggest that MT-III at 37°C binds to and penetrates the plasma membrane of cultured myoblasts, thereby inducing a rapid, direct and irreversible membrane permeabilization. This effect apparently depends in part on the PLA2 activity of the toxin and in part on a molecular region which is separate from the catalytic site.
