On the mode of action of basic phospholipase A2 from Naja nigricollis venom

A study has been made of the cytotoxic activity of basic phospholipase A2 of venom from Naja nigricollis on different types of cells and of the participation of esterase activity in this cytotoxic activity. It was previously shown that the cytotoxicity observed is not due to a contaminant, since the cytotoxic action vanished after immunoprecipitation of basic phospholipase A2 by specific monoclonal antibodies. All eukaryotic cells tested were sensitive to the cytotoxic action of basic phospholipase A2. In contrast, Escherichia coli K-12 wild strain was resistant. Participation of cell membranes in the sensitive or resistant character of cells to the phospholipase A2 attack was investigated using E. coli K-12 membrane mutants. Some membrane mutants were sensitive and the sensitivity or resistance to basic phospholipase A2 was found to be related to a single mutation in the locus envA. The requirement for esterase activity of phospholipase A2 in cytotoxic attack has been shown to be dependent on the cell line tested. Indeed, when the esterase activity of basic phospholipase A2 was eliminated by treatment with p-bromophenacyl bromide, the enzyme retained a cytotoxic potency inducing necrosis of certain tumoral cells grown in vitro, but not other cells such as erythrocytes, for which concomitant esterase activity was also necessary. In vivo studies of toxicity showed that the loss of either cytotoxic potency or esterase activity eliminates the lethal character of basic phospholipase A2. This leads us to propose that in vivo toxicity of phospholipase A2 depends on simultaneous expression of esterase activity and a non-enzymatic property, manifested by in vitro cytotoxic potency.