Proposal for molecular mechanism of thionins deduced from physico-chemical studies of plant toxins.

We propose a molecular model for phospholipid membrane lysis by the ubiquitous plant toxins called thionins. Membrane lysis constitutes the first major effect exerted by these toxins that initiates a cascade of cytoplasmic events leading to cell death. X-ray crystallography, solution nuclear magnetic resonance (NMR) studies, small angle X-ray scattering and fluorescence spectroscopy provide evidence for the mechanism of membrane lysis. In the crystal structures of two thionins in the family, alpha(1)- and beta-purothionins (MW: approximately 4.8 kDa), a phosphate ion and a glycerol molecule are modeled bound to the protein. (31)P NMR experiments on the desalted toxins confirm phosphate-ion binding in solution. Evidence also comes from phospholipid partition experiments with radiolabeled toxins and with fluorescent phospholipids. This data permit a model of the phospholipid-protein complex to be built. Further, NMR experiments, one-dimensional (1D)- and two-dimensional (2D)-total correlation spectroscopy (TOCSY), carried out on the model compounds glycerol-3-phosphate (G3P) and short chain phospholipids, supported the predicted mode of phospholipid binding. The toxins' high positive charge, which renders them extremely soluble (>300 mg/mL), and the phospholipid-binding specificity suggest the toxin-membrane interaction is mediated by binding to patches of negatively charged phospholipids [phosphatidic acid (PA) or phosphatidyl serine (PS)] and their subsequent withdrawal. The formation of proteolipid complexes causes solubilization of the membrane and its lysis. The model suggests that the oligomerization may play a role in toxin's activation process and provides insight into the structural principles of protein-membrane interactions.

Binding to and hemolysis of human erythrocytes by pyrularia thionin and Naja naja kaouthia cardiotoxin: inhibition by prothrombin.

Pyrularia thionin and snake venom cardiotoxin are strongly basic proteins which bind to and induce hemolysis of erythrocytes, cause depolarization of muscle cells, and influence the order and properties of phospholipids in cellular membranes. Earlier studies showed a competition between the thionin and cardiotoxin for a common binding site on erythrocytes, and the present study extends these studies to show a similar competition between prothrombin and both basic proteins. The competition between the thionin and prothrombin for binding sites on erythrocytes was shown by direct binding experiments using radiolabeled thionin. Whereas binding of thionin or cardiotoxin induces hemolysis as a consequence of membrane perturbation, prothrombin does not induce hemolysis. Although it binds to the same site, there is no penetration into and perturbation of the membrane. The competition between prothrombin and pyrularia thionin is not influenced by added Ca++. This indicates that membrane PS interacts in a specific and Ca++-independent manner with at least one site on prothrombin, as proposed earlier (Tendian and Lentz, 1990). The competition between thionin and prothrombin was also demonstrated for the release of radiolabeled chromate from loaded P388 cells. The competition observed with the P388 cells shows that the competition is not a unique phenomenon with erythrocytes, but occurs with other cell membranes.

Interactions of thionin from Pyrularia pubera with dipalmitoylphosphatidylglycerol large unilamellar vesicles.

The peptide toxin thionin from Pyrularia pubera binds to dipalmitoylphosphatidylglycerol (DPPG) large unilamellar vesicles as shown by an increase in the intensity and blue-shift of the fluorescence emission spectrum of the single tryptophan residue of the protein. The magnitude of these fluorescence changes increased with temperature near the thermotropic phase transition of DPPG (about 40 degrees C). Fluorescent probes sensitive to the structure and dynamics of the membrane were used to assess the effect of thionin binding on bilayer properties. The fluorescence emission spectra of Prodan, Patman, and Laurdan all showed spectral changes consistent with an increase in bilayer polarity at temperatures below the DPPG phase transition but a decrease in polarity at higher temperatures. Fluorescence polarization experiments and the ratio of monomer-to-excimer fluorescence of the probe 1,3-bis(1-pyrene)propane suggested that thionin increases the bilayer order above the transition temperature. Differential scanning calorimetry revealed that thionin broadens the transition and either increases or decreases the melting temperature depending on the concentration of the peptide. Taken together, the data are consistent with at least three distinct interactions of thionin with the bilayer: (1) thionin bound electrostatically to the bilayer surface; (2) tryptophan of the bound thionin inserted into the bilayer; (3) high-order aggregates of thionin-bound vesicles.

Mechanisms by which thionin induces susceptibility of S49 cell membranes to extracellular phospholipase A2.

Whereas cells normally resist attack by PLA2, they become susceptible under certain pathological conditions. To ascertain the regulatory mechanisms that induce cellular susceptibility to PLA2, the effect of thionin on S49 cells was examined in the presence of PLA2. Thionin alone was unable to evoke hydrolysis of the lipid bilayer. Likewise, the addition of PLA2 alone caused production of only a minimal amount of free fatty acid. However, thionin and PLA2 together resulted in significant hydrolysis of the cell membrane. Thionin caused perturbation of the bilayer structure as suggested by the changes in the emission spectra of laurdan and the permeability of the membrane to propidium iodide. These changes correlated quantitatively with the susceptibility of the lipid bilayer to PLA2. Furthermore, thionin induced a modest increase in intracellular Ca2+. The source of this Ca2+ was the extracellular fluid since EDTA in the extracellular medium inhibited the Ca2+ influx. Moreover, cobalt chloride, a universal Ca2+ channel blocker, prevented the rise in intracellular Ca2+, the uptake of propidium iodide, and the susceptibility to PLA2 induced by thionin. In contrast, the changes in the laurdan emission caused by the thionin were not affected by the cobalt. Furthermore, incubation of the cells with the calcium ionophore A23187 also caused the cells to become susceptible to PLA2. We hypothesize that thionin causes S49 cell membranes to become susceptible to PLA2 by a Ca2+-dependent perturbation of the bilayer structure.

In vitro evaluation of Pyrularia thionin-anti-CD5 immunotoxin.

The membrane-active peptide, Pyrularia thionin, purified from Pyrularia pubera, was covalently conjugated to an anti-CD5 monoclonal antibody. The membrane-active properties of thionin were not affected by the conjugation. The immunotoxin killed CD5+ lymphocytes in vitro at a concentration of 0.1 nmol/10(7) cells after 2 h of incubation. The immunotoxin also inhibited the proliferation of T cells in vitro, stimulated either by mitogens or in the mixed lymphocyte reaction. It was shown by electron paramagnetic resonance of spin probes and differential scanning calorimetry that the ability of the immunotoxin to perturb the lipid phase of membranes is close to that of unconjugated thionin. The results obtained suggest that Pyrularia-thionin-anti-CD5 conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5+ leukemia and lymphomas.

Modulation of phospholipase A2 activity by membrane-active peptides on liposomes of different phospholipid composition.

To determine the influence of variations in both lipid species and lipid packing on phospholipase A2 (PLA2) hydrolytic activity, the activities of two PLA2 isolated from Crotalus molossus molossus venom, were followed on unilamellar liposomes modified by membrane-active peptides. Enzymatic activity was compared with cytolytic activity on human and mouse lymphocytes. Phosphatidylcholine liposomes were hydrolysed better than liposomes containing acidic phospholipids (phosphatidylserine, phosphatidic acid or cardiolipin) or phosphatidylethanolamine. Both membrane-active peptides, cardiotoxin and thionin, inhibited the PLA2 activity on phosphatidylcholine liposomes. The activities of the enzymes were profoundly enhanced on thionin-pretreated liposomes containing phosphatidylserine, and on cardiotoxin-pretreated liposomes containing cardiolipin or phosphatidic acid. Both cardiotoxin and thionin facilitated the cytolytic activities of PLA2 on both human and mouse lymphocytes. Cytolytic activity correlated well with esterase activity. It is proposed that the complex dynamic structure of cell membranes renders a variety of substrate configurations that transiently affect PLA2 activity.

Enhancement of adenylate cyclase activity in S49 lymphoma cell membranes by the toxin thionin from Pyrularia pubera.

We investigated the mode of action of Pyrularia thionin on adenylate cyclase in S49 lymphoma cell membranes. Thionin increased the forskolin-induced cAMP production in both wild-type and cyc- variant cell membranes. Thionin increased the apparent efficacy, but not potency, of the stimulation of adenylate cyclase by forskolin. Our results also indicated that the stimulatory G protein (Gs) was not involved in the action of thionin on adenylate cyclase; however, thionin appeared to reduce the effect of Gi on adenylate cyclase. We examined the effect of thionin on the temperature dependence of adenylate cyclase activity. The effect of thionin was not quantitatively the same at all temperatures. The effect was greatest in the range of about 30 degrees C to 50 degrees C. In addition, cardiotoxin, a structurally distinct peptide that alters the properties of biological membranes similarly to thionin, also enhanced the activity of adenylate cyclase. It seems reasonable to suspect that thionin influences the activity of adenylate cyclase indirectly by acting on the membrane.

Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes.

Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.

Modification of phospholipid membrane structure by the plant toxic peptide Pyrularia thionin.

Pyrularia thionin (P thionin) is a bioactive peptide from the parasitic plant Pyrularia pubera. The structural aspects of its interaction with phospholipid membranes were investigated by measuring the responses of phosphorescence quenching, EPR spin labels, and 1H and 31P NMR at different phospholipid compositions. In phosphatidylcholine bilayers containing cardiolipin or phosphatidylinositol, P thionin induced a pronounced increase in the membrane viscosity, and at higher P thionin concentrations the formation of nonbilayer structures was observed. In phosphatidylcholine bilayers containing phosphatidylserine, P thionin induced a significant transformation of membrane lamellar structure accompanied by a decrease of membrane viscosity. In all investigated lipid systems added P thionin caused an increase in membrane permeability and induced a fusion of sonicated liposomes. The specificity of P thionin interaction with phosphatidylserine-containing membrane is discussed and a model of phospholipid membrane modification by P thionin is suggested.

Pyrularia thionin increases arachidonate liberation and prolactin and growth hormone release from anterior pituitary cells.

Pyrularia thionin is a 47 amino acid peptide isolated from the nuts of Pyrularia pubera. This peptide does not have intrinsic phospholipase A2 activity, but it increases the liberation of arachidonate from several tissues. Exposure of anterior pituitary cells to this toxin increases the liberation of arachidonate, increases the cellular levels of lysophospholipids, and decreases cellular phospholipids. Thus, phospholipase A2 is involved in the liberation of arachidonate stimulated by this peptide. Because this toxin also increases stearate liberation from the pituitary cells, either diacylglycerol lipase, phospholipase A1 or lysophospholipase may be directly or indirectly activated by this toxin. In addition to increasing fatty acid liberation, Pyrularia thionin increases the release of prolactin and growth hormone from anterior pituitary cells over the identical concentration ranges that this toxin liberates the fatty acids. Pyrularia thionin increased arachidonate liberation and prolactin release from perifused pituitary cells within 2 min, and following withdrawal of the toxin, arachidonate liberation and prolactin release returned to near basal levels within 6 min. Dopamine, a physiological inhibitor of prolactin release that closes calcium channels, decreased prolactin release stimulated by Pyrularia thionin. However, dopamine had no effect on the arachidonate liberation stimulated by this peptide. Similarly, D-600, an organic calcium channel blocker, decreased the prolactin and growth hormone release stimulated by the toxin without affecting the toxin-stimulated arachidonate liberation. Therefore, Pyrularia thionin increases arachidonate liberation through the rapid activation of phospholipase A2 by a mechanism that is not dependent on calcium uptake via D-600-inhibitable calcium channels. In contrast, the prolactin and growth hormone release stimulated by this toxin requires calcium uptake via D-600 inhibitable calcium channels.

Role of Tyr and Trp in membrane responses of Pyrularia thionin determined by optical and NMR spectra following Tyr iodination and Trp modification.

Pyrularia thionin is a strongly basic and bioactive 47 amino acid peptide which contains two Tyr residues at positions 13 and 45 and one Trp at position 8. Limited iodination does not have a significant effect, but prolonged iodination of the peptide leads to progressive inactivation for all known cellular responses (Evans, J. et al. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5849-5853). 1H NMR spectra of the native Pyrularia thionin show four Tyr bands, two arising from each Tyr residue. One resonance band for the epsilon hydrogens of Tyr 45 disappears early during limited iodination and the band from the delta hydrogens shifts to low field. The two bands corresponding to Tyr 13 remain during limited iodination, but both decrease in intensity during prolonged iodination, with the epsilon hydrogen band decreasing somewhat more. The resonance bands arising from Trp disappear during prolonged iodination. This sequence of reactions is verified by the optical absorbance properties of two small peptide fragments obtained by Staphylococcal V8 protease hydrolysis of thionin which had been iodinated to varying degrees. Limited iodination did not significantly inhibit the thionin's biological activity, yet the fragment from the -COOH terminus showed the conversion of Tyr 45 to diiodoTyr. This treatment did not significantly modify the Tyr 13 or Trp 8 located in the -NH2 terminal fragment. More extensive iodination resulted in a disappearance of Trp 8 absorbance with an accompanying conversion of Tyr 13 to the monoiodo form. Extensive iodination yielded two atoms of iodine in the Tyr 45-containing fragment, and only one atom in the Tyr 13 fragment. The data indicate that Tyr 45 of the native thionin is more readily iodinated, proceeding to the diiodo form without significant loss of activity. Prolonged iodination does not lead to the formation of any diiodoTyr 13, but does lead to modification of Trp 8 and probably formation of monoiodoTyr 13. Modification of Trp 8 with N-bromosuccinimide inhibits the hemolytic activity of the thionin, showing that Trp 8 is necessary for Pyrularia thionin activity. It is most likely Trp 8 modification during prolonged iodination which results in the loss of biological activity.

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

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 A2 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 Ca2+, and by removing Ca2+ 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 Ca2+ 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 Ca2+ in the 5-10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.

Binding properties of Pyrularia thionin and Naja naja kaouthia cardiotoxin to human and animal erythrocytes and to murine P388 cells.

Pyrularia thionin and snake venom cardiotoxin are strongly basic peptides which induce hemolysis, depolarization of muscle cells and activation of endogenous phospholipase A2. An earlier study of the hemolysis reaction indicated that the two peptides bind to and compete for the same site on human erythrocytes. A recent study examined the hemolysis induced by both peptides as the phosphate and Ca2+ content of the reaction mixture was varied. The results of the recent study (VERNON, L. P. and ROGERS, A., Toxicon 30, 701-709) agree with this companion study on the binding of 125I-labeled pyrularia thionin and cardiotoxin to erythrocytes under the same conditions. Added phosphate ion at 5 mM and removal of membrane-bound Ca2+ by treatment with 10 mM EGTA make more binding sites of the same affinity available to both peptides, which are shown to bind in a competitive fashion to the same site. Addition of 10 mM Ca2+ to the medium decreases peptide binding due to competitive binding of Ca2+ to the same site on the membrane. For human erythrocytes the number of binding sites/cell for the thionin ranged from 0.7 to 1.7 x 10(5) and for cardiotoxin from 0.82 to 1.6 x 10(5). The calculated dissociation constants (Kd) from the Scatchard plots ranged from 0.43 to 1.1 microM for the thionin and from 0.40 to 0.98 microM for the cardiotoxin. The binding sites for thionin and cardiotoxin with sheep erythrocytes were 1.7 and 2.0 x 10(4) sites/cell, respectively, and both cow and horse erythrocytes demonstrated 2.7 x 10(4) sites/cell for the thionin. Binding studies with murine P388 cells showed 7.0 and 9.5 x 10(6) sites per cell for Pyrularia thionin and cardiotoxin, respectively.

Membrane structure, toxins and phospholipase A2 activity.

The phospholipid-hydrolyzing enzyme phospholipase A2 (PLA2) (EC 3.1.1.4) exists in several forms which can be located in the cytosol or on cellular membranes. We review briefly cellular regulatory mechanisms involving covalent modification by protein kinase C and the action of Ca2+, cytokines, G proteins and other cellular proteins. The major focus is the role of phospholipid structure on PLA2 activity, including (1) the mechanism of PLA2 action on synthetic phospholipid bilayers, (2) perturbation of synthetic and cellular membranes with lipophilic agents and membrane-interactive peptides and (3) the ability of these agents to activate endogenous PLA2 activity, with emphasis on the venom and plant toxins melittin, cardiotoxin and Pyrularia thionein.

Hemolysis of erythrocytes and fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols and benzylidene derivatives.

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.

Developmental stages of cucumber seedlings: kinetics of chlorophyll accumulation and other growth parameters.

The changes in morphology during dark germination and subsequent growth of cucumber (Cucumis sativus) seedlings in the light go through three different phases described as latent, active, and steady-state. This pattern is consistently observed for several related developmental processes. The latent period lasts about 2 days following water imbibition after which the following capabilities appear in concert: (a) root and stem elongation, (b) pigment synthesis including protochlorophyll, chlorophyll, carotenoid, and phytochrome, (c) synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase, and (d) enhancement of greening by excision. Following the active phase, which lasts for another 2 to 3 days, these processes slow to a steady-state. Inhibition of chlorphyll accumulation by SO(2) was only observed for seedlings in the steady-state phase.

Phospholipase activation in the cytotoxic mechanism of thionin purified from nuts of Pyrularia pubera.

Treating NIH3T3 fibroblast cells with Pyrularia pubera thionin (100 micrograms/ml) stimulated release of labelled free fatty acids from phospholipids biosynthetically labelled by incorporation of [3H]arachidonic acid. Since Pyrularia thionin exhibited no detectable phospholipase activity, it was concluded that the release response represented activation of endogenous phospholipases in the cells. The phospholipase activated by Pyrularia thionin (100 micrograms/ml) stimulated the release of 61% of the incorporated [3H]arachidonate in the presence of 1.8 mM extracellular calcium with maximum activation at 90 min following an initial lag period of about 20 min. The release response exhibited little dependence on extracellular calcium at this thionin concentration, but at concentrations 20 micrograms/ml, extracellular calcium appeared to be inhibitory to phospholipase activation. Some characteristics of the fatty acid release response are consistent with activation of a lysosomal phospholipase being part of the cellular response to Pyrularia thionin. Activation of a lysosomal enzyme can occur independently or as a result of coordinate activation of the whole lysosome, which would expose other cellular components of degradative lysosomal enzymes. Consistent with coordinate activation of lysosomal enzymes, Pyrularia thionin also stimulates the production of small, trichloroacetic acid-soluble peptides and nucleic acid fragments from biosynthetically-labelled RNA and proteins in treated cells. It is not clear from the results obtained what role, if any, activation of lysosomal enzymes plays in the overall toxic response to Pyrularia thionin in NIH3T3 cells. However, Pyrularia pubera thionin may represent a useful tool for studying the regulation of lysosomal enzymes and their roles in cells.

Conformational energy analysis of the pentapeptide Ac-Arg-Asn-Cys-Tyr-Asn-NMA from alpha 1-purothionin.

Conformational energy analyses were carried out on the pentapeptide RNCYN (Ac-Arg-Asn-Cys-Tyr-Asn-NMA) and on related peptides. RNCYN is a highly conserved amino acid sequence in thionins and viscotoxins. The conformation of the pentapeptide was calculated to be an amphipathic alpha-helix, with the tyrosine and cysteine on the nonpolar side of the helix and the arginine and both asparagines on the polar side. Our results are inconsistent with the conformation determined using the Chou-Fasman prediction method, but are consistent with the conformation determined experimentally (using n.m.r.) for this pentapeptide sequence in alpha 1-purothionin.

Cellular responses to Pyrularia thionin are mediated by Ca2+ influx and phospholipase A2 activation and are inhibited by thionin tyrosine iodination.

Pyrularia thionin, isolated from nuts of Pyrularia pubera, is a strongly basic peptide of 47 amino acids. The amino acid sequence and configuration of its four disulfide bonds place this plant peptide, known to be hemolytic, cytotoxic, and neurotoxic, among the thionins. We report and compare several cellular responses mediated by Pyrularia thionin: hemolysis of human erythrocytes, activation of an endogenous phospholipase A2 in Swiss 3T3 cells, cytotoxicity toward HeLa and mouse B16 melanoma cells in culture, viability of rat hepatocytes and lymphocytes measured by trypan blue exclusion, and lethality in mice. Cellular responses related to ion movement include a toxin-mediated influx of Ca2+ into mouse P388 cells measured by Fura-2 fluorescence, depolarization of mouse P388 plasma membrane measured by fluorescence of bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol), and depolarization of frog (Rana pipiens) sartorius muscle determined by direct measurement of membrane potential. Graded iodination of Pyrularia thionin leads to a related loss of activity for hemolysis, phospholipase A2 activation, cytotoxicity, and lethality in mice. The mediated Ca2+ influx into and depolarization of P388 cells require Ca2+ in the external medium and are inhibited by 100 microM Ni2+. Depolarization of sartorius muscle by Pyrularia thionin also requires a functional Ca2+ channel, as shown by verapamil inhibition. This muscle depolarization also involves phospholipase A2 activation because dexamethasone and quinacrin, but not indomethacin, protect against depolarization. The IC50 values for viability of rat hepatocytes and splenic lymphocytes measured by trypan blue exclusion were 0.17 and 40 microM, respectively. The general response of cells to Pyrularia thionin involves a membrane alteration leading to depolarization and a channel-mediated influx of Ca2+. There is a related activation of phospholipase A2 that results in loss of membrane integrity, hemolysis in the case of erythrocytes, and eventually cell death. Iodination of Pyrularia thionin leads to a corresponding inhibition of all three cellular responses, which indicates an essential role for tyrosine in either maintenance of peptide structure or interaction of the peptide with cellular membranes.