Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent $\text{p}\text{K}$ of the terminal carboxyl group of Lys-Trp-Lys is raised by ～3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.     

Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.     

Clavanin permeabilizes target membranes via two distinctly different pH-dependent mechanisms

The pH dependence of the antimicrobial and membrane activity of clavanin A, a peptide antibiotic that is rich in histidines and glycines, was analyzed in growth and membrane leakage experiments. Clavanin A more effectively inhibited the growth of the test organism Lactobacillus sake when the pH of the medium was lowered. Whereas the wild-type peptide efficiently released fluorophores from unilamellar vesicles at neutral pH according to a nonspecific permeabilization mechanism, it did not permeabilize model bilayers at low pH. It was therefore suggested that this peptide uses a distinct mode of action under acidic conditions different than that used around neutral pH. However, at low pH, the membrane is still the target for clavanin A, as the peptide collapsed both vital transmembrane proton gradients and ion gradients under these conditions. Clavanin A did not act as a ionophore across phospholipid bilayers, indicating that membrane constituents other than membrane phospholipids are involved in the dissipation of transmembrane ion gradients. Membrane proteins that generate transmembrane ion gradients are suggested to be the targets for clavanin A at low pH. In addition to the histidines, the three glycine residues of clavanin A are shown to play an important role in the specific mode of interaction with these membrane targets. These residues may induce a flexible hydrophobic conformation that allows the peptide to exert different membrane activities. This study demonstrates that clavanin A is a special membrane-active peptide that has access to two markedly distinct pH-dependent modes of actions.     

Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis

A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.     

Membrane fusion induced by mutual interaction of the two charge-reversed amphiphilic peptides at neutral pH

An anionic amphiphilic peptide and the charge-reversed cationic peptide are synthesized. They contain 20 amino acids with the same sequence except for 5 Glu residues for the anionic versus 5 Lys residues for the cationic peptides. Fusion of egg phosphatidylcholine large unilamellar vesicles is assayed with the fluorescent probes by the lipid mixing and the internal content mixing at neutral pH. The peptide mixture causes a rapid and efficient membrane fusion, in spite of no fusions with each peptide by itself. Each peptide takes nearly random coils with a small amount of helix, but the peptide mixture has an ordered helical structure. The equimolar peptide mixture forms a much more hydrophobic complex than those of different molar ratios of peptides and also that of each peptide itself. The equimolar peptide mixture causes the most efficient fusion. Preincubations of two peptides before addition to vesicles cause the slower rates of fusion. The fusion is greatly reduced at higher ionic strength and nearly zero at 800 mM NaCl and 40 mM sodium phosphate. Each peptide and the peptide mixture show the same alpha-helical structure, interact with vesicles, but do not induce fusion at higher ionic strengths. These results suggest that the two peptides interact mutually through the electrostatic Coulombic interaction between the charged groups. The electrically neutralized hydrophobic complex aggregates the separate vesicles together and interacts with the hydrocarbon region of lipid bilayers to cause fusion.     

Conformational transitions of membrane-bound HIV-1 fusion peptide

The human immunodeficiency virus type-1 (HIV-1) fusion peptide (FP) functions as a non-constitutive membrane anchor that translocates into membranes during envelope glycoprotein-induced fusion. Here, by means of infrared spectroscopy (IR) and of various bilayer-perturbation assays, we describe the peptide conformations that are accessible to its membrane-bound state and the transitions occurring between them. The peptide underwent a conformational transition from a predominantly α-helical structure to extended β-type strands by increasing peptide concentration in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) vesicles. A comparable transition was observed at a fixed 1:100 peptide-to-lipid ratio when calcium was added to vesicles containing prebound α-helical peptide. Cation binding induced an increase in the amount of H-bonded carbonyls within the interfacial region of POPG. Calcium-promoted α→β conversion in membranes correlated with the closure of preformed lytic pores and took place in dispersed (nonaggregated) vesicles doped with poly(ethylene glycol)-lipid conjugates, showing that the conformational transition was independent of vesicle aggregation. We conclude that the target membrane conditions modulate the eventual structure adopted by the HIV-1 FP. Conformational polymorphism of the inserted peptide may contribute to the flexibility of the fusogenic complex during the fusion reaction cycle, and/or may be related to target membrane perturbation at the fusion locus.     

Conformational transitions of membrane-bound HIV-1 fusion peptide

The human immunodeficiency virus type-1 (HIV-1) fusion peptide (FP) functions as a non-constitutive membrane anchor that translocates into membranes during envelope glycoprotein-induced fusion. Here, by means of infrared spectroscopy (IR) and of various bilayer-perturbation assays, we describe the peptide conformations that are accessible to its membrane-bound state and the transitions occurring between them. The peptide underwent a conformational transition from a predominantly alpha-helical structure to extended beta-type strands by increasing peptide concentration in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) vesicles. A comparable transition was observed at a fixed 1:100 peptide-to-lipid ratio when calcium was added to vesicles containing prebound alpha-helical peptide. Cation binding induced an increase in the amount of H-bonded carbonyls within the interfacial region of POPG. Calcium-promoted alpha-->beta conversion in membranes correlated with the closure of preformed lytic pores and took place in dispersed (nonaggregated) vesicles doped with poly(ethylene glycol)-lipid conjugates, showing that the conformational transition was independent of vesicle aggregation. We conclude that the target membrane conditions modulate the eventual structure adopted by the HIV-1 FP. Conformational polymorphism of the inserted peptide may contribute to the flexibility of the fusogenic complex during the fusion reaction cycle, and/or may be related to target membrane perturbation at the fusion locus.     

Effect of phospholipid composition on an amphipathic peptide-mediated pore formation in bilayer vesicles

To better understand the influence of phospholipid acyl-chain composition on the formation of pores by cytotoxic amphipathic helices in biological membranes, the leakage of aqueous contents induced by the synthetic peptide GALA (WEAALAEALAE ALAEHLAEALAEALEALAA) from large unilamellar phospholipid vesicles of various compositions has been studied. Peptide-mediated leakage was examined at pH 5.0 from vesicles made of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with the following acyl-chain compositions: 1-palmitoyl-2-oleoyl (PO), 1,2-dioleoyl (DO), 1, 2-dielaidoyl (DE), and 1,2-dipetroselinoyl (DPe). A mathematical model predicts and simulates the final extents of GALA-mediated leakage of 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) from 1-palmitoyl-2-oleoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phospha tidylglycerol (POPC/POPG) and 1, 2-dielaidoyl-sn-glycero-3-phosphocholine/1, 2-dielaidoyl-phosphatidylglycerol (DEPC/DEPG) liposomes at pH 5.0 as a function of peptide concentration in the bilayer, by considering that GALA pores responsible for this leakage have a minimum size of 10 +/- 2 monomers and are formed by quasiirreversible aggregation of the peptide. With the phospholipid acyl-chain compositions tested, GALA-induced ANTS/DPX leakage follows the rank order POPC/POPG approximately DEPC/DEPG > DPePC/DPePG > DOPC/DOPG. Results from binding experiments reveal that this reduced leakage from DOPC/DOPG vesicles cannot be explained by a reduced binding affinity of the peptide to these membranes. As shown by monitoring the leakage of a fluorescent dextran, an increase in the minimum pore size also does not explain the reduction in ANTS/DPX leakage. The data suggest that surface-associated GALA monomers or aggregates are stabilized in bilayers composed of phospholipids containing a cis unsaturation per acyl chain (DO and DPe), while transbilayer peptide insertion is reduced. GALA-induced ANTS/DPX leakage is also decreased when the vesicles contain phosphatidylethanolamine (PE). This lends further support to the suggestion that factors stabilizing the surface state of the peptide reduce its insertion and subsequent pore formation in the bilayer.     

Determination of the hydrophobic binding site of phosphatidylcholine exchange protein with photosensitive phosphatidylcholine.

1-Acyl-2-(7-(4-azido-2-nitrophenoxy)-[1-14C]heptanoly)-sn-glycero-3-phosphocholine was synthesized in order to study the lipid-binding site of the phosphatidylcholine exchange protein from bovine liver. Photosensitive phosphatidylcholine was incorporated into the protein by incubation with vesicles of this phosphatidylcholine derivative. The lipid-protein complex was separated from the vesicles by chromatography on Biogel A-0.5m. Photolysis of the complex by irradiation with light of a high pressure mercury lamp at a wavelength above 340 nm generated the highly reactive nitrene. Sodium dodecyl sulfate gel electrophoresis of the photolysed complex indicated that 30% of the endogenous 14C-labeled phosphatidylcholine was covalently linked to the protein. Peptides were isolated after digestion of the photolysed complex with protease from Staphylococcus aureus and trypsin. It was determined that the 2-acyl chain of the phosphatidylcholine molecule was linked to the peptide segment -Gly-Ser-Lys-Val-Phe-Met-Tyr-Tyr-. This segment was part of a protease peptide of about 65 residues of which the sequence was determined by Edman degradation for the first 38 residues. This peptide contains a cluster of apolar residues -Val-Phe-Met-Tyr-Tyr-Phe with an extremely high hydrophobicity index and with a predicted beta-sheet conformation. It is concluded that this hydrophobic cluster forms part of the binding site.     

Dynorphin induced magnetic ordering in lipid bilayers as studied by P NMR spectroscopy

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T_m=24°C), as examined by ³¹P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T_m but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T_m. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T_m in parallel with the bilayer surface, because a single ³¹P NMR signal appeared at the perpendicular position of the ³¹P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T_m, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated ³¹P NMR lineshape.     

The heterogeneity of bound acetylcholine and synaptic vesicles

Synaptic vesicles containing radioactive acetylcholine have been isolated from slices of Torpedo electric organ incubated with radioactive choline. The recently synthesized radioactive acetylcholine is preferentially removed from the vesicles by iso-osmotic gel filtration. There is therefore a small compartment of loosely bound recently synthesized acetylcholine within the monodisperse vesicle fraction. The specific radioactivity of this compartment correlates most closely with the ;free' acetylcholine of electric organ that is lost when the tissue is homogenized. Membrane-associated vesicles did not contain any particular enrichment of this compartment. On standing at 6 degrees C the loosely bound compartment stabilizes so that it survives iso-osmotic filtration. A study of this phenomenon revealed that it was proportional to the extent of the loss of tightly bound acetylcholine from the vesicles. Incubation with Ca(2+), at pH5.5, or partial hypo-osmotic shock, caused losses of tightly bound acetylcholine and proportional increases in the stabilization of loosely bound acetylcholine of vesicles. Incubation at 20 degrees C caused less loss of tightly bound, and less stabilization of loosely bound, acetylcholine. A theoretical treatment of these exchanges also shows that the random factors promoting loss of tightly bound acetylcholine are statistically correlated with those which cause stabilization of loosely bound acetylcholine. The reciprocal relationship between the exchanges is inconsistent with there being two distinct populations of vesicles, one containing recently synthesized, loosely bound acetylcholine and the other containing tightly bound acetylcholine. It is proposed that all the vesicles contain a core of tightly bound acetylcholine and a surface layer of loosely bound acetylcholine. The origin of the extravesicular acetylcholine and also of the acetylcholine released on stimulation is discussed in the light of these results.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Apocytochrome c induces pH-dependent vesicle fusion

The ability of apocytochrome c and the heme containing respiratory chain component, cytochrome c, to induce fusion of phosphatidylcholine (PC) small unilamellar vesicles containing 0-50 mol % negatively charged lipids was examined. Both molecules mediated fusion of phosphatidylserine (PS):PC 1:1 vesicles as measured by energy transfer changes between fluorescent lipid probes in a concentration- and pH-dependent manner, although cytochrome c was less potent and interacted over a more limited pH range than the apocytochrome c. Maximal fusion occurred at pH 3, far below the pKa of the 19 lysine groups contained in the protein (pI = 10.5). A similar pH dependence was observed for vesicles containing 50 mol % cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylinositol (PI) in PC but the apparent pKa values varied somewhat. In the absence of vesicles, the secondary structure of apocytochrome c was unchanged over this pH range, but in the presence of negatively charged vesicles, the polypeptide underwent a marked conformational change from random coil to alpha-helix. By comparing the pH dependencies of fusion induced by poly-L-lysine and apocytochrome c, we concluded that the pH dependence derived from changes in the net charge on both the vesicles and apocytochrome c. Aggregation could occur under conditions where fusion was imperceptible. Fusion increased with increasing mole ratio of PS. Apocytochrome c did induce some fusion of vesicles composed only of PC with a maximum effect at pH 4. Biosynthesis of cytochrome c involves translocation of apocytochrome c from the cytosol across the outer mitochondrial membrane to the outer mitochondrial space where the heme group is attached. The ability of apocytochrome c to induce fusion of both PS-containing and PC-only vesicles may reflect characteristics of protein/membrane interaction that pertain to its biological translocation.     

Hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, induced permeability change of phosphatidylcholine bilayers

Interactions of hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, with phosphatidylcholine vesicles were investigated to obtain information on its bioactive mechanism. The peptide induced the leakage of a fluorescent dye, calcein, entrapped in sonicated vesicles. The leakage rate depended on both the peptide and the lipid concentrations. Analysis of this dependency indicated that the leakage was due to the monomeric peptide and that the membrane-perturbing activity of the monomer was higher for solid distearoylphosphatidylcholine vesicles than for fluid egg yolk phosphatidylcholine vesicles. Hypelcin A also affected the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The transition was broadened with a reduced transition enthalpy, suggesting the peptide strongly binds the surrounding lipids to perturb the bilayer lipid packing. A circular dichroism study revealed that the helical content of hypelcin A increases upon membrane binding. We concluded that the monomeric peptide with an increased helical content, complexed with the lipids, perturbs the lipid organization and induces the increased permeability.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Effect of water soluble non-membrane proteins on the phospholipid structure

The interaction of water-soluble nonmembraneous proteins (trypsin and the basic pancreatic trypsin inhibitor (BPTI)) with soybean phospholipids was studied using multilamellar vesicles. Multilamellar vesicles were obtained from soybean lipid extracts and mixtures of individual phospholipids based on phosphatidylcholine. These mixtures contain different phospholipids: "bilayer", "nonbilayer", and negatively charged. It was shown that the content of both proteins in the complex depends on pH and the presence of negatively charged components. On the basis of this finding, the conclusion about the electrostatic nature of lipid-protein interaction was made. The structural organization of soybean phospholipids in multilamellar vesicles was studied in the presence and absence of the proteins using broad-line 31P-NMR spectroscopy. It was found that, in mixtures of phospholipids of complex composition, different types of phases coexist, and phospholipids of different classes can compensate the effects of each other. Trypsin and BPTI affect the structure of phospholipids in a similar way, inducing considerable structural changes in multilamellar vesicles of preparations containing negatively charged components in whose structure there coexisted primordially the bilayer and isotropic phases.     

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.