Effect of calcium and phosphate ions on hemolysis induced by Pyrularia thionin and Naja naja kaouthia cardiotoxin

Abstract

Pyrularia thionin is a strongly basic bioactive peptide of 47 amino acids isolated from nuts of Pyrularia pubera. It is hemolytic, cytotoxic and activates an endogenous phospholipase A 2 in 3T3 cells. Earlier studies have shown that the cardiotoxin from Naja naja kaouthia has similar activities and binds to the same site as Pyrularia thionin. Since the peptides appear to bind to the phospholipids of cell membranes to elicit their cellular responses, the effect of modifying the electrostatic environment was studied by separately adding phosphate ion and Ca 2+, and by removing Ca 2+ from the membrane by treatment with EGTA. Analysis of erythrocyte hemolysis for both Pyrularia thionin and cardiotoxin shows that the reactions follow Michaelis-Menten kinetics, with the peptides serving as the substrate. The basal rate of hemolysis in physiological saline is markedly increased by the addition of phosphate in the 5–10 mM range and also by removing membrane-bound Ca 2+ by incubation of the cells with 10 mM EGTA. These treatments do not change the apparent K m values, but increase the V max, indicating that more binding sites are made available by these treatments. On the other hand, added Ca 2+ in the 5–10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.