Interaction of wasp venom mastoparan with biomembranes

Mastoparan-induced changes in the K+ permeability of rat peritoneal mast cells, human erythrocytes, Staphylococcus aureus and Escherichia coli were examined. Mastoparan did not efficiently increase the K+ permeability of cells except for S. aureus. The release of membrane phospholipids was also observed from S. aureus cells in the concentration range of the permeability enhancement. Mastoparan stimulated histamine release from mast cells, independently of a small efflux of K+. Mastoparan became markedly effective to E. coli cells whose outer membrane structure was chemically disrupted beforehand, showing that the peptide can enhance the permeability of the cytoplasmic membranes of both Gram-positive and -negative bacteria. In experiments using liposomes, mastoparan increased the permeability of the liposomes composed of egg phosphatidylethanolamine and egg phosphatidylglycerol, which are the lipid constituents of the cytoplasmic membrane of E. coli cells, while it showed a weak activity to the liposomes composed of egg phosphatidylcholine and cholesterol. The latter result related closely to the fact that this peptide acted weakly on erythrocytes and mast cells in which acidic lipids constitute a minor portion. Mastoparan decreased the phase transition temperature of dipalmitoylphosphatidylglycerol liposomes, but it did not affect that of dipalmitoylphosphatidylcholine liposomes. These results indicate that mastoparan penetrated into membranes mainly containing acidic phospholipids and disrupted the membrane structure to increase the permeability. The action of the wasp venom mastoparan was compared with that of a bee venom melittin.     

Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57-63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.     

Mode of action of gramicidin S on Escherichia coli membrane

The action of a cationic antibiotic gramicidin S on the outer and cytoplasmic membranes of Escherichia coli was studied. It was found that gramicidin S disrupted the permeability barrier of the outer membrane, permitting the permeation of an antibiotic ionophore, this being similar to the action of the dimer in compound 48/80 (Katsu, T., Shibata, M. and Fujita, Y. (1985) Biochim. Biophys. Acta 818, 61-66). However, differently from the dimer, gramicidin S further stimulated the efflux of K+ through the cytoplasmic membrane of E. coli. The time course of K+ permeability change accorded well with that of change in the viability of E. coli cells. These changes occurred at temperatures above the phase transition of the cytoplasmic membrane. This temperature range differed greatly from the case of polymyxin B, a polycationic antibiotic acting at temperatures above the phase transition of the outer membrane. We discuss the mode of gramicidin S action on the cytoplasmic membrane of E. coli, in comparison with the results on red blood cells and liposomes.     

Ion channels of various types induced in lipid membranes by gramicidin A derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta6.3 single-stranded and beta5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Comparative study of the membrane-permeabilizing activities of mastoparans and related histamine-releasing agents in bacteria, erythrocytes, and mast cells

The membrane-permeabilizing activities of mastoparans and related histamine-releasing agents were compared through measurements of K(+) efflux from bacteria, erythrocytes, and mast cells. Changes in bacterial cell viability, hemolysis, and histamine release, as well as in the shape of erythrocytes were also investigated. The compounds tested were mastoparans (HR1, a mastoparan from Polistes jadwagae, and a mastoparan from Vespula lewisii), granuliberin R, mast cell-degranulating peptide, and compound 48/80, as well as antimicrobial peptides, such as magainin I, magainin II, gramicidin S, and melittin. We used a K(+)-selective electrode to determine changes in the permeability to K(+) of the cytoplasmic membranes of cells. Consistent with the surface of mast cells becoming negatively charged during histamine release, due to the translocation of phosphatidylserine to the outer leaflet of the cytoplasmic membrane, histamine-releasing agents induced K(+) efflux from mast cells, dependent on their ability to increase the permeability of bacterial cytoplasmic membranes rich in negatively charged phospholipids. The present results demonstrated that amphiphilic peptides, possessing both histamine-releasing and antimicrobial capabilities, induced the permeabilization of the cytoplasmic membranes of not only bacteria but mast cells. Mastoparans increased the permeability of membranes in human erythrocytes at higher concentrations, and changed the normal discoid shape to a crenated form. The structural requirement for making the crenated form was determined using compound 48/80 and its constituents (monomer, dimer, and trimer), changing systematically the number of cationic charges of the molecules.     

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.     

The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. The role of induced proton permeability

1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin–dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria.     

Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.     

Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli.

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.     

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

Plicatamide,an antimicrobial octapeptide from Styela plicata hemocytes

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc Delta DOPA), where dc Delta DOPA represents decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dc Delta DOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design.     

Role of mitochondrial outer membrane in the uncoupling activity of N-terminally glutamate-substituted gramicidin A

Of a series of gramicidin A (gA) derivatives, we have earlier found the peptide [Glu1]gA exhibiting very low toxicity toward mammalian cells, although dissipating mitochondrial membrane potential with almost the same efficiency as gA. Substitution of glutamate for valine at position 1 of the gA amino acid sequence, which is supposed to interfere with the formation of ion-conducting gA channels via head-to-head dimerization, reduces both channel-forming potency of the peptide in planar lipid bilayer membranes and its photonophoric activity in unilamellar liposomes. Here, we compared [Glu1]gA and gA abilities to cause depolarization of the inner mitochondrial membrane in mitochondria and mitoplasts, the latter lacking the outer mitochondrial membrane. Importantly, much less gA was needed to decrease the membrane potential in mitoplasts than in mitochondria, whereas the depolarizing potency of [Glu1]gA was nearly the same in these systems. Moreover, in multilamellar liposomes, [Glu1]gA exhibited more pronounced protonophoric activity than gA, in contrast to the data for unilamellar liposomes. These results allowed us to conclude that [Glu1]gA has a much higher permeability between adjacent lipid membranes than gA. Therefore, the fraction of peptide molecules, reaching the inner mitochondrial membrane upon the addition to cells, is much higher for [Glu1]gA compared to gА. Under these conditions, the decreased cytotoxicity of [Glu1]gA could be associated with its low efficiency as a channel-former dissipating potassium and sodium ion gradients across plasma membrane. The present study highlighted the role of the ability to permeate among various biological membranes for intracellular efficiency of ionophores.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Effective lipid‐detergent system for study of membrane active peptides in fluid liposomes

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X‐100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X‐100 occurs (R_e^sat), was determined by light scattering. Measurements of steady‐state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X‐100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at R_e^sat was studied by CD spectroscopy. It was found that the conformational state of this channel‐forming peptide changed crucially when Triton X‐100 induced transition to more fluid membranes. The gA single‐channel measurements were made with Triton X‐100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid‐detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane‐active peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

家蝇幼虫抗菌肽MDL-2的电泳制备及其抗菌作用机理

通过体壁损伤和感染大肠杆菌同时诱导家蝇Musca domestica幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收,复性,抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-2,其分子中富含Pro,Gly和碱性氨基酸,分子量为11 kD,对革兰氏阴性菌Escherichia coli和革兰氏阳性菌Staphylococcus aureus均有较强抗性,因此电泳制备抗菌肽的方法为此类生物微量活性物质的分离纯化提供一种行之有效的途径。通过MDL-2对大肠杆菌和金黄色葡萄球菌通透性和透射电镜超微结构的图谱分析,MDL-2首先与细菌的外膜结合,然后抗菌肽形成柔性的两亲空间构象与细胞内膜作用,扰乱了膜脂分子的排列,改变了细胞膜的通透性,影响细胞膜的结构和功能,细胞膜上形成了许多孔道,同时造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

Dication and trication which can increase the permeability of Escherichia coli outer membrane

Recent success in the preparation of the monomer, dimer and trimer in compound 48/80 prompted us to investigate the action of these compounds on Escherichia coli cells. It was found that compound 48/80 inhibited growth of E. coli cells, while the monomer, dimer and trimer in 48/80 did not. However, the following experiments showed that the dimer and trimer disrupted the permeability barrier of the outer membrane of E. coli. First, addition of the dimer or trimer in cell suspension stimulated the uptake of tetraphenylphosphonium cation. Second, the synergistic effect of the dimer on the action of gramicidin caused the efflux of K+. In experiments using isolated cytoplasmic membrane vesicles, addition of gramicidin alone caused the efflux of K+. Thus, it was speculated that, with whole cells, the dimer formed some defect structure in the outer membrane, through which gramicidin reached the cytoplasmic membrane and increased the K+ permeability. The temperature dependence of efflux K+ showed that the dimer in 48/80 rendered the outer membrane permeable to gramicidin at temperatures above the phase transition of the outer membrane.     

Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces

Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.     

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.     

Effect of hypoglycemic sulfonylureas on Ca2+ fluxes across lipid bilayers

Black lipid membranes and liposomes loaded with Ca2+ or 5,6-carboxyfluorescein were used for exploring the mechanism of action of insulin-releasing sulfonylureas. Unlike the Ca2+/H+ exchanging ionophore A-23187, tolbutamide did not stimulate the net efflux of Ca2+ from the liposomes. Glibenclamide caused a sustained release of Ca2+, but this effect could be attributed to labilization of the liposomal membrane as indicated by a quantitatively similar loss of the stability marker 5,6-carboxyfluorescein. Unlike the neutral ionophore nonactin or the channel forming quasi-ionophore gramicidin A, the sulfonylureas did not alter the conductance of black lipid membranes in medium containing Na+, K+, Ca2+, Mg2+, and Cl-. It is concluded that the sulfonylureas tested lack ionophore properties but that glibenclamide can labilize membranes.