Isolation, sequence, and conformation of seven trichorzianines B from Trichoderma harzianum

From the antagonistic fungus Trichoderma harzianum, a group of acidic new peptides, trichorzianines B (TB), was isolated in addition to neutral trichorzianines A (TA) previously studied. TA and TB exhibit various biological activities related to their membrane properties and a different behaviour of the two groups was noticed. As observed for other peptaibols, TB consist in a microheterogeneous mixture which was resolved into pure peptides by reversed-phase C18 HPLC. The sequence of the seven main isolated TB, namely TB IIa, TB IIIc, TB IVb, TB Vb, TB VIa, TB VIb, TB VII, was determined by the combined use of positive ion FAB mass spectrometry and 2D 1H n.m.r. spectroscopy, including COSY and NOESY experiments. TB differ from the corresponding TA only by the replacement of Gln 18 in the TA sequence by a glutamic acid. The 1H n.m.r. data suggested that the TB are mainly organized in an alpha helix.     

Prevention of nonspecific lysis in liposomal and erythrocyte immunoassay systems by small lipid vesicles and erythrocyte ghosts

Large unilamellar liposomes prepared by the reverse-phase evaporation method (REVs) were made immunoreactive by incorporating dinitrophenylaminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) or 8-(3-carboxypropyl)-theophylline-dipalmitoylphosphatidylethanolamine (Th-DPPE) into the phospholipid bilayer. Specific lysis in the presence of anti-DNP-BSA and goat anti-theophylline serum respectively, was induced by adding guinea pig serum as source for complement to these liposomes. However, specific lysis was found to be compromised by high levels of nonspecific lysis as monitored by the release of the fluorescent aqueous-space marker 6-carboxyfluorescein. Nonspecific lysis could be prevented without affecting specific lysis by pretreatment of complement or incubation of the reaction mixture with small unilamellar liposomes (SUVs). SUVs of various lipid compositions produced the desired effect; however, when the fraction of negative charge in the SUVs was increased to 30 mol%, specific lysis was inhibited as well. In a similar assay system consisting of hemolysin-sensitized sheep red blood cells it was also found that nonspecific lysis could be inhibited by addition of erythrocyte ghosts to the incubation medium, although specific lysis was somewhat depressed. However, SUVs or REVs of a composition similar to sheep erythrocytes were ineffective indicating a more selective nature of complement-mediated immunoreaction with erythrocyte membranes than with synthetic bilayer membranes.     

Studies on the interaction of tiamulin with the phospholipids in model membranes and with microsomes.

The drug tiamulin interacts with phospholipid membranes mainly in a nonelectrostatic way. At pH-values where the drug possesses a net positive charge only small binding is observed. In the presence of cholesterol tiamulin is excluded from the membranes. The interaction of tiamulin with membranes cannot be explained by a simple partitioning but is governed by structural rearrangements of the lipid phase. At low drug concentrations we observe sigmoidal binding characteristics in the rigid as well as in the fluid state up to a level of about 2-3 mol drug bound per 1000 mol phospholipid. The range in which this cooperative interaction occurs can be compared with the drug concentration in the erythrocyte membrane which protects from hypotonic lysis. Further addition of tiamulin to the rigid membrane leads to fluidization. Saturation of the membranes with tiamulin is completely in parallel to their fluidization. The relevance of the cooperative interaction at low drug concentration and of the subsequent fluidization at elevated concentration for the microsomal membrane is discussed.     

Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.     

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.     

Permeability properties of unilamellar vesicles containing choline plasmalogens and comparison with other choline glycerophospholipid species

The rates of non-electrolyte and ion diffusion across bilayer membranes consisting of choline plasmologens or of their alkyl and acyl analogs were studied. The influx of [¹⁴C]glucose, ⁸⁶Rb⁺ and ³⁶Cl^? into small unilamellar vesicles made from a semisynthetic choline plasmalogen and from synthetic diacyl, alkylacyl and dialkyl analogs with comparable side chain compositions were measured. Rates of glucose and Rb⁺ diffusion are about equal in alkenylacyl- and diacyl-glycerophosphocholine (GPC) bilayers, but are reduced in dialkyl-GPC membranes; the permeability coefficients correlate with the packing densities of the respective choline glycerophospholipids in monolayers at the air water interface. Rates of chloride diffusion are consistently higher in membranes formed from phospholipids containing alkenyl or alkyl other bonds as compared to the diacyl analogs. Highest rates of Cl^? diffusion are observed with choline plasmalogen vesicles. The phospholipid side chain composition has little influence on Cl^? permeation, but glucose and Rb⁺ diffusion are markedly affected. Incorporation of cholesterol (30 mol%) into choline plasmalogen membranes reduces their solute permeability by approximately 70%. A similar effect is found with the other choline phospholipid analogs. Thus, the choline phospholipid—cholesterol interaction, as far as it is reflected in reduced bilayer permeability, is not influenced by the presence of the alkenylether bond of plasmalogens.     

Interaction of the Latrodectus tredecimguttatus spider venom with liposomes

It is established that interaction of Latrodectus tredecimguttatus venom with bilayer phospholipid membranes of liposomes results in formation of ion channels. Liposome permeability variations are estimated by input and output of 86Rb+ and 45Ca2+ isotopes. It is shown that LaCl3 in 0.5 mM concentration decreases considerably both input and output of Rb+ in proteoliposomes and it may be used as an inhibitor of latrotoxin channels in experiments with phospholipid vesicles. When studying electrophoretic spectrum of proteins bound with liposomes it is shown that latrotoxin (a polypeptide) with molecular mass 130 kDa predominates in proteoliposomes.     

Site of action of the local anesthetic tetracaine in a phosphatidylcholine bilayer with incorporated cardiolipin.

Tetracaine (TTC) increases the permeability of phospholipid liposomal membranes to water, and this increase is reduced by the incorporation of cardiolipin into the membranes. We examined the molecular interaction of a phospholipid with the TTC cation in egg-yolk phosphatidylcholine (EyPC) liposomal membranes with incorporated bovine heart cardiolipin (BhCL) by IR spectroscopy and by determination of partitioning and the pKa of membrane-bound TTC. The IR spectra indicated that TTC shifted the stretching band of the BhCL PO2- group, a potential site of hydration in the bilayer, to a lower frequency but did not shift that of EyPC. TTC intercalated into the BhCL bilayer shifted its aromatic C-N stretching band to a lower frequency. One molecule of TTC was found to bind approximately five molecules of EyPC, and the incorporation of negatively charged BhCL into EyPC membranes increased the degree of binding of TTC to the bilayer membranes. The pKa values of TTC bound to membranes were determined as 7.7, 9.4, and 10.2 for EyPC membranes, EyPC membranes containing 50 mol % BhCL, and BhCL membranes, respectively, whereas that in an aqueous 10-mM NaCl solution was 8.5, as it was dependent on the manner of binding. The IR data together with the partitioning and the pKa data suggested differences between the actions of the TTC cation on negatively charged BhCL and on neutrally charged EyPC polar groups in the region close to the aqueous interface of the lipid bilayer.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes.

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Effect of fatty acids and monoglycerides on permeability of lipid bilayer

The effect of fatty acids and monoglycerides on barrier properties of liposomal membranes prepared from egg phosphatidylcholine was investigated. The incorporation of these lipids as liposomal membrane components induced the alteration of the permeability to less permeable liposomally entrapped drugs, sulfanilic acid and procainamide ethobromide (PAEB). Monoolein caused greatly increased permeability of both drugs and unsaturated fatty acids markedly enhanced the release rate of PAEB, while saturated fatty acids caused a small increase in the release rate.Electron spin resonance (ESR) investigation with 5-nitroxide stearic acid showed that fatty acids disordered the hydrophobic region of the lipid bilayer and the disordering effect of unsaturated fatty acids was greater than that of saturated ones. It was demonstrated that the incorporated fatty acids and monoglycerides interacted with the polar region of the membranes by ESR study with cholestane label and ¹H-NMR study. These results indicated that the increase in the membrane permeability caused by fatty acids and monoglycerides associated with the disorder in the membranes' interior and the interaction of the incorporated lipid with the polar head group of phospholipid.     

Effect of chain length of HAV-VP3 synthetic peptides on its interaction with biomembrane models

Shorter analogues of a continuous epitope of hepatitis A virus, VP3(110-121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. To better understand the influence of the structural properties of this 12-mer peptide epitope on its biological activity, the interaction of smaller peptide analogues with phospholipid biomembrane models was investigated by a combination of spectroscopic and biophysical techniques. In this article we describe our findings concerning the surface activity and the interaction of peptides with simple mono- and bilayer membranes composed of a zwitterionic phospholipid (dipalmitoyl phosphatidylcholine, DPPC), an anionic phospholipid (dipalmitoyl phosphatidylglicerol, DPPG), or a DPPC/DPPG mixture. The results indicate that the net negative charge of the peptide is in some way responsible of the specific interactions between VP3(110-121) and membrane phospholipids, and necessary to induce beta-type conformations upon vesicle interaction.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic α-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Interaction of enkephalin peptides with anionic model membranes

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen²,D-Pen⁵]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.