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 A2; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A2; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a Km of 1.6 microM; (5) binding studies with 125I-thionin show by Scatchard analysis a Kd value of 2.1 microM; (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, Ki, for this reaction is 7.0 microM; and (7) Ca2+ 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 A2. The release of fatty acids is itself not sufficient to explain the hemolytic reaction.
Osorio e Castro
V R
VR
Department of Chemistry, Pontificia Universidade Catolica, Rio de Janeiro, Brasil.
Van Kuiken
B A
BA
Vernon
L P
LP
D023181:Antimicrobial Cationic Peptides; D002118:Calcium / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects*; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009754:Nuts / Q000032:analysis*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000633:toxicity*
D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; C044942:THI1 protein, Pyrularia pubera; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium