Hemolytic activity of thionin from Pyrularia pubera nuts and snake venom toxins of Naja naja species: Pyrularia thionin and snake venom cardiotoxin compete for the same membrane site

Abstract

— Pyrularia thionin ( P. thionin) is a strongly basic peptide of 47 amino acids which is hemolytic, cytotoxic and neurotoxic. It shows the greatest hemolytic activity toward human erythrocytes. Rabbit, guinea pig and pig erythrocytes show decreasing activity in that order, and little or no activity is shown with sheep, horse, cow or mouse erythrocytes. Crotalus venoms are inactive, but the venoms from Naja naja atra, Naja naja ceylonicus and Naja naja melanoleuca and, more specifically, cardiotoxin from Naja naja kaouthia have significant hemolytic activities toward human erythrocytes. The cardiotoxin preparation used had no phospholipase activity, and was less active than P. thionin (23% compared to 35% hemolysis by P. thionin in 60 min at 10 μg/ml toxin). Since iodinated P. thionin is inactive, it was used as an inhibitor of hemolysis catalyzed by native P. thionin, N. ceylonicus venom and by cardiotoxin. Examination of the kinetics of the reactions catalyzed by N. ceylonicus venom and cardiotoxin in the absence and presence of iodinated P. thionin shows that both N. ceylonicus venom and cardiotoxin exhibit Michaelis-Menten kinetics, yielding apparent K m values of 7.4 μg/ml and 0.69 μM, respectively. These values compare to an apparent K m for P. thionin of 1.6 μM for erythrocyte hemolysis and a binding constant of 2.1 μM ( Osorio e Castro, V. R., van Kuiken, B. A. and Vernon, L. P. (1989) Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes. Toxicon 27, 501). The inhibition constants K i for iodinated P. thionin in the reactions with N. ceylonicus venom and cardiotoxin are 3.8 and 5.3 μM, respectively. These compare to a K i value of 7.0 μM for the reaction catalyzed by native P. thionin. These data point to similar mechanisms of action of cardiotoxin and P. thionin on cellular membranes, including a common binding site which could be a protein in the membrane.