The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes.

The effects of temperature and of the membrane-active protein CTII on the formation of nonbilayer structures in mitochondrial membranes were studied by 31P-NMR. Increasing the temperature of isolated mitochondrial fractions correlated with an increase in ATP synthase activity and the formation of nonbilayer packed phospholipids with immobilized molecular mobility. Computer modeling was employed for analyzing the interaction of mitochondrial membrane phospholipids with the molecular surface of CTII, which behaves like a dicyclohexylcarbodiimide-binding protein (DCCD-BP) of the F0 group in a lipid phase. Overall, our studies suggest that proton permeability toroidal pores formed in mitochondrial membranes consist of immobilized nonbilayer-packed phospholipids formed via interactions with DCCD-BP. Our studies support the existence of a proton transport along a concentration gradient mediated via transit toroidal permeability pores which induce conformational changes necessary for mediating the catalytic activity of ATP synthase in the subunits of the F0-F1 complex.

Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom.

The venom composition of Mojave rattlesnakes (Crotalus scutulatus scutulatus) differs in that some individuals have Mojave toxin and others do not. In order to understand the genetic basis for this difference, genomic DNA samples from Mojave rattlesnakes collected in Arizona, New Mexico, and Texas were analyzed for the presence of DNA sequences that relate to the acidic (Mta) and basic (Mtb) subunits of this toxin. DNA samples were subjected to PCR to amplify nucleotide sequences from second to fourth exons of the acidic and basic subunits. These nucleotide sequences were cloned and sequenced. The nucleotide sequences generated aligned exactly to previously published nucleotide sequences of Mojave toxin. All DNA samples analyzed generated product using the basic subunit primers, and aligned identically to the Mtb nucleotide sequence. However, only 11 out of the 14 samples generated a product with the acidic subunit primers. These 11 sequences aligned identically to the Mta nucleotide sequence. The venom from the three snakes whose DNA did not amplify with the acidic subunit primers were not recognized by antibodies to Mojave toxin. This suggests that snakes with venom lacking Mojave toxin also lack the productive nucleotide sequence for the acidic subunit in their DNA.

Cobra venom cytotoxin free of phospholipase A2 and its effect on model membranes and T leukemia cells.

Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine (PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat cells and normal lymphocytes were killed equivalently when treated with 10(-9) m PLA2 and 10(-5) m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.

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.

Phospholipase A2 and cobra venom cytotoxin Vc5 interactions and membrane structure.

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.

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.

[Mitochondrial proteolipids]. / Proteolipidy mitokhondrii.

A convenient procedure is proposed for extracting mitochondrial proteolipids using a single phase mixture chloroform-methanol-water (1:2:0.8 v/v) with subsequent separation of the phases. The proteolipids were concentrated at the interface between the phases and thus purified from the bulk of the phospholipids. It was found that the mitochondrial proteolipids represent stable complexes of phospholipids with some low molecular weight proteins (M(r) = 7-18 kDa). The latter are destroyed at acid pH values. The phospholipid/protein ratio was found to be equal to 6 (assuming the molecular masses of the proteins and phospholipids to be equal to 10 and 0.8 kDa, respectively). The phospholipid composition of the tightly bound proteolipids thus obtained did not differ from that of the mitochondrial phospholipids. Using 31P-NMR, nonbilayer structures were found to arise from proteolipid reconstitution into multibilayer liposomes.

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.

[Intermembrane exchange of lipids induced by cobra venom cytotoxins]. / Mezhmembrannyi obmen lipidami, indutsiruemyi tsitoksinami iada kobry.

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.

[The fusogenic properties of the cytotoxins of cobra venom in a model membrane system]. / Issledovanie f'iuzogennykh svoistv tsitotoksinov iada kobry v sisteme model'nykh membran.

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.

Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes.

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.