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Pyrularia thionin (PT) is a basic 47 amino acid peptide isolated from the nuts of Pyrularia pubera. Its structure and properties have been studied in some detail. Its receptor site is a domain of membrane phosphatidyl serine (PS), where it binds with a relatively high specificity. A segment of its covalent structure, the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, designated serine nonapeptide (SNP), corresponds to amino acids 7-15 of the thionin, except for the position 12 (Ser), which substitutes for Cys, to give stability. This peptide represents what we consider to be the active site of the thionin, and it also binds to PS domains, but less tightly than thionin does. The peptide has an effect on the prothrombinase assay using the chromophore S2238 to measure the thrombin produced by the prothrombinase complex. It is shown that SNP stimulates the prothrombinase complex activity, instead of inhibiting it, as would be expected if it simply covered the PS sites on the membrane of erythrocyte ghosts, used in the prothrombinase assay. SNP appears to substitute for Va in the prothrombinase complex reaction, in a Ca(2+) independent manner, being even more effective in the absence than in the presence of ghosts. In the clotting system, SNP can also substitute for Factor Va.

Hemolysis rates of human erythrocytes induced by C2 and C8-C14 straight chain 1-alkanols, 1,2-alkanediols and the corresponding benzylidene derivatives (benzaldehyde acetals) have been studied and compared with hemolysis rates obtained by three peptide toxins. The peak of activity occurs at C12 for the alkanols and glycols and at C10 for the benzylidene derivatives. The most active compound is 1-dodecanol, followed by 1,2-dodecanediol and the C10 benzylidene acetal, which show 50% hemolysis at 15, 99 and 151 microM, respectively, at 37 degrees C. A few lysolecithins and longer chain cis-unsaturated alcohols were studied for comparison purposes, and were found to be more active than 1-dodecanol. The most active were the 16:0 lysolecithin and cis-9-tetradecene-1-ol, which gave 50% hemolysis at concentrations of 2.8 and 5.6 microM respectively. The hemolytic activities of 1-dodecanol, 1,2-dodecanediol and the C10 benzylidene acetal were compared to activities of Pyrularia thionin and melittin with cow, horse, sheep, pig and human erythrocytes. Whereas the peptide toxins showed clear specificity for human erythrocytes, no selectivity was shown by any of the other compounds tested. Addition of the thionin or Naja naja kaouthia cardiotoxin to erythrocyte ghosts caused a slight but reproducible increase in the order of the phospholipid bilayer, as measured with the fluorescent probe NBD-PC. Cardiotoxin gave a greater response than did the P thionin, and extensively iodinated P thionin gave a smaller change than did P thionin. Similar results were obtained with melittin, but this peptide gave a markedly greater response than all other peptides. Addition of dodecanol or the C10 benzylidene acetal caused a marked increase in membrane fluidity. All of these data indicate that the organic compounds interact directly with and are incorporated nonspecifically into the membrane lipid bilayer, but the peptide toxins interact specifically with some component on the surface of the membrane, either a protein or specific phospholipid domain, followed by insertion into the membrane and decreasing phospholipid movement.

— 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 125 I-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.

—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 Km values of 7.4 μg/ml and 0.69 μM, respectively. These values compare to an apparent Km 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. Toxicon27, 501). The inhibition constants Ki 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 Ki 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.