EFFECTS ON CULTURED-MAMMALIAN-CELLS OF MYOTOXIN-III, A PHOSPHOLIPASE-A(2) ISOLATED FROM BOTHROPS-ASPER (TERCIOPELO) VENOM

Myotoxin III (MT-III), a myotoxic phospholipase A2 from Bothrops asper , was studied with respect to interactions with cultured mammalian cel ls and red blood cells. Tests of the cytopathogenic effect of MT-III o n different cell lines indicated that rat skeletal muscle L6 myoblasts were more sensitive to the toxin than chinese hamster ovary cells, hu man lung fibroblasts, mouse adrenal tumour cells and rat intestinal ep ithelial cells. Specific plasma-membrane permeabilization was assayed as release of a cytosolic [H-3]uridine nucleotide marker from toxin-tr eated L6 cells. A dose- and time-related membrane permeabilization was induced at 37-degrees-C, but not at 0-degrees-C. A half-maximal effec t was obtained after 20 min. 30 mug/ml MT-III induced 50% marker relea se in 1 h, and the effect was not reversed by post-incubation for up t o 48 h in toxin-free medium. The membrane permeabilization in L6 cells did not seem to require cellular internalisation of the toxin. The ca talytic site of the toxin was inactivated by alkylation with p-bromoph enacyl bromide (BPB). This treatment abolished the toxin's specific PL A2 activity, as assayed in vitro, and reduced the PLA2 activity on the myoblast membrane by more than 95%, as measured by release of [C-14]a rachidonic acid from prelabelled cells. However, the membrane-permeabi lizing effect (release of cytosolic marker) was reduced only by 70% up on modification with BPB. We also report that MT-III is not directly h aemolytic, and one reason for this is the inability of the toxin to as sociate with the membranes of human or mouse erythrocytes. Taken toget her, the data suggest that MT-III at 37-degrees-C binds to and penetra tes the plasma membrane of cultured myoblasts, thereby inducing a rapi d, direct and irreversible membrane permeabilization. This effect appa rently depends in part on the PLA, activity of the toxin and in part o n a molecular region which is separate from the catalytic site.