Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes

— Pyrularia thionin is a strongly basic peptide of 47 amino acids isolated from Pyrularia pubera. This peptide, a member of the thionin family, is hemolytic, cytotoxic and neurotoxic. The characteristics of the hemolytic activity on human erythrocytes are as follows: (1) the peptide does not itself have any phospholipase activity in a micellar assay system with egg yolk phosphatidylcholine, as evidenced by a lack of pH change or uptake of oxygen in the presence of lipoxidase; (2) erythrocyte membranes treated with thionin, however, show a low level of oxygen uptake in the presence of lipoxidase as a consequence of fatty acid release, and this activity is synergistic with that of bee venom phospholipase A 2; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A 2; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a K m of 1.6 μM; (5) binding studies with 125I-thionin show by Scatchard analysis a K d value of 2.1 μM; (6) although iodinated thionin is inactive in the hemolysis assay, it acts as a competitive inhibitor to native thionin in the hemolytic assay; the inhibitor constant, K i, for this reaction is 7.0 μM; and (7) Ca 2+ above 1 mM inhibits the reaction. All the data are consistent with thionin binding to a receptor, most likely a protein, on the erythrocyte membrane, leading to the release of free fatty acids, most likely by activation of phospholipase A 2. The release of fatty acids is itself not sufficient to explain the hemolytic reaction.