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Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

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.

The hydrolytic activity and interaction of acidic and neutral phospholipase A2 (PLA2) with large unilamellar liposomes treated with cobra venom cytotoxin Vc5 (CT Vc5) were studied to more fully understand the modulating effects of cationic membrane-active peptides on PLA2. Studies were done by fluorescence displacement, EPR spin probes, and 31P-NMR. The results showed that CT Vc5 inhibits PLA2 activity on phosphatidylcholine liposomes. Enzymatic activity of both acidic and neutral PLA2's were enhanced on liposomes containing cardiolipin and pretreated with cytotoxin. The cytotoxin, however, inhibited enzyme lipid hydrolysis if these same liposomes were first treated with acidic PLA2. The highest enzymatic activity was found on substrates with nonbilayer lipid packing. Using EPR of spin labeled enzymes, it was shown that CT Vc5 inhibited binding of acidic PLA2 to liposomes and caused displacement of acidic PLA2 from liposomes. No direct interaction was found between CT Vc5 and neutral PLA2.. It is suggested that cytotoxin perturbs packing of lipid molecules in liposomes containing cardiolipin and is responsible for increased catalysis, whereas direct interaction between CT Vc5 and acidic PLA2, inhibits enzyme activity. It is concluded that variability in substrate composition and the chemical nature of both PLA2 and cationic peptide determine whether enzyme activity is affected by substrate packing or by direct enzyme-peptide interaction. Models of interactions of PLA2 with CT Vc5 and phospholipid membranes are presented.

Interaction between cytotoxin Vc5 from cobra venom and model membranes from phosphatidylcholine and phosphatidyl acid was investigated by means of 1H-NMR, ESR spin probes and microcalorimetric methods. Cytotoxic was shown to induce in the membrane formation of non-bilayer structures, to increase the membrane permeability and to produce the intermembrane exchange with lipids.

By the methods of EPR spinal probes, energy migration of triplet excitation and NMR spectroscopy, the structural changes on hydrocarbon region of membranes, the changes in dynamic state of water of lipid hydrate jacket, the intermembrane lipid material exchange and fusion of membranes induced by cytotoxins of cobra venom have been studied. The sequence of events preceded the membrane fusion is suggested. The probability of membrane fusion has been shown not to be determined by fusogenic agent structure only, but much it depends on lipid composition of membranes.

The processes of membrane aggregation, permeability and fusion induced by cytotoxins from Central Asian cobra venom were investigated by studying optical density of liposome samples, permeability of liposome membranes for ferricyanide anions and exchange of lipid material between the membranes of adjacent liposomes. Cytotoxins Vc5 and Vc1 were found to induce aggregation of PC + CL and PC + PS liposomes. Cytotoxin Vc5 increased also the permeability of the liposomes for K3[Fe(CN)6] and enhanced their fusion. Cytotoxin Vc1 increased membrane permeability and enhanced fusion of PC + CL samples only. The changes in membrane permeability and fusion were found to occur within a single value of cytotoxin concentrations. The fusogenic properties of the cytotoxins studied are supposed to be due to the ability to dehydrate membrane surface and to destabilize the lipid bilayer structure. Fusion probability is largely defined by the phospholipid composition of the membranes. A model of interaction of cytotoxins with cardiolipin-containing membranes is offered.