Effect of tryptophan oligopeptides on the size distribution of POPC liposomes: a dynamic light scattering and turbidimetric study

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.     

Increased phospholipase a(2) activity with phosphorylation of peroxiredoxin 6 requires a conformational change in the protein

We have shown previously and confirmed in this study that the phospholipase A(2) (PLA(2)) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluates the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, strengthened ANS binding, and a lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ΔG°(D) was 3.3 ± 0.3 kcal mol(-1) for Prdx6 and 1.7 ± 0.7 kcal mol(-1) for pPrdx6, suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (50:25:15:10 DPPC:egg PC:cholesterol:PG molar ratio) were evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with a higher affinity (K(d) = 5.6 ± 1.2 μM) than Prdx6 (K(d) = 24.9 ± 4.5 μM). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (ΔH = -31.49 ± 0.22 kcal mol(-1)). Correlating our conformational studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA(2) activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA(2) enzymatic activity.     

Interaction of aldolase A with lecithin liposomes

The aldolase A binding to the lecithin liposomes (Kd = 2.4 +/- 0.1 X 10(-3) M) has been shown by the fluorescence and tryptophan phosphorescence at the room temperature. The interaction is accompanied by an increase in the phospholipid bilayer microviscosity, and some conformational changes in the hydrophobic part of the enzyme, pronouncing themselves in Trp-147 environment rigidity, decrease. The observation of membrane viscosity vs. incubation time revealed practically instant enzyme-membrane interaction and no gradual incorporation. The accessibility of the NAD-binding domain of aldolase for NADH in the liposome presence remains unaltered.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of alkylphospholipid liposomes with MT-3 breast-cancer cells depends critically on cholesterol concentration

We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposome formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells.     

Membrane-active properties of alpha-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides.

Reaction of the melanotropin hormone analogs [Nle(4),D-Phe(7)]-alpha-MSH and [Nle(4),D-Phe(7)]-alpha-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle(4),D-Phe(7)]-alpha-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of alpha-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a beta-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

Clumping factor B, a fibrinogen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesin of Staphylococcus aureus, also binds to the tail region of type I cytokeratin 10

The primary habitat of Staphylococcus aureus in humans is the moist squamous epithelium of the anterior nares. We showed previously that S. aureus adheres to desquamated epithelial cells and that clumping factor B (ClfB), a surface-located MSCRAMM (microbial surface components recognizing adhesive matrix molecules) known for its ability to bind to the alpha-chain of fibrinogen, is partly responsible (O'Brien, L. M., Walsh, E. J., Massey, R. C., Peacock, S. J., and Foster, T. J. (2002) Cell. Microbiol. 4, 759-770). We identified cytokeratin 10 (K10) as the ligand recognized by ClfB. Here we have shown that purified recombinant human and murine K10 immobilized on a plastic surface supports adherence of S. aureus in a ClfB-dependent manner. Furthermore, the recombinant A domain of ClfB (rClfB 45-542) bound to immobilized K10 dose-dependently and saturably. Subdomains of human and murine K10 were expressed and purified. The N-terminal head domain (residues 1-145) did not support the binding of rClfB or adherence of S. aureus ClfB+. In contrast, the C-terminal tail domains (human rHK10 452-593, mouse rMK10 454-570) promoted avid binding and adherence. Isothermal titration microcalorimetry and intrinsic tryptophan fluorescence experiments gave dissociation constants for rClfB 45-542 binding to rMK10 454-570 of 1.4 and 1.7 microM, respectively. The tail region of K10 is composed largely of quasi-repeats of Tyr-(Gly/Ser)n. A synthetic peptide corresponding to a typical glycine loop (YGGGSSGGGSSGGY; Y-Y loop peptide) inhibited the adherence of S. aureus ClfB+ to immobilized MK10 to a level of 80%, whereas control peptides had no effect. The KD of rClfB 45-542 for the Y-Y loop peptide was 5.3 microm by intrinsic tryptophan fluorescence. Thus ClfB binds to the glycine loop region of the tail domain of keratin 10 where there are probably multiple binding sites. Binding is discussed in the context of the dock-lock-latch model for MSCRAMM-ligand interactions. We provide an explanation for the molecular basis for S. aureus adherence to the squamous epithelium and suggest that nasal colonization might be prevented by reagents that inhibit this interaction.     

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: Cholesterol acyltransferase: An energy transfer study

• 1.1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin: cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions.
• 2.2. In the absence of lipids no fluorescence energy transfer was measurable.
• 3.3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface.
• 4.4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.

     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0-250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (D(c) = 80 +/- 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O(2) removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A(0)) increases in O(2)-free buffer (98 +/- 13) and in 10% ethanol-containing buffer (74 +/- 34) compared to control conditions (23 +/- 5). Varying the dose rate between 1-10 Gy/min at a constant dose of 50 Gy, an inverse dose-rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. II: Biotinylated liposomes and antibiotin antibody

The aggregation of biotinylated phospholipid vesicles (liposomes) cross-linked by antibiotin IgG was studied experimentally and theoretically. The liposomes were either low density liposomes that contained 0.4 mol% biotinylated phospholipid ( approximately 100 exposed biotin molecules per liposome), or high density liposomes that contained 2.7 mol% biotinylated phospholipid ( approximately 1000 exposed biotin molecules per liposome). The solution turbidity and mean particle size measured by quasi-elastic light scattering (QLS) were monitored throughout the aggregation. Three different lots of antibiotin antibodies, each with different association constants and binding heterogeneities, were used. The antibody binding characteristics affected the aggregation rates. The aggregation kinetics were analyzed using a model based on the Smoluchowski theory of aggregation, fractal concepts of aggregate microstructure, and Rayleigh and Mie light scattering theory. The experimental conditions of liposome concentration, protein concentration, and ligand density under which aggregation occurred correlated well with calculated sticking probabilities based on isotherms describing the adsorption of antibiotin antibody to the liposomes. These results are compared with prior observations made when avidin was used as the cross-linking protein. (c) 1996 John Wiley & Sons, Inc.     

Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.     

Membrane features and activity of GPI-anchored enzymes: alkaline phosphatase reconstituted in model membranes

The influence of membrane lipid environment on the activity of GPI-anchored enzymes was investigated with human placental alkaline phosphatase reconstituted by a detergent-dialysis technique in liposomes composed of palmitoyloleoylphosphatidylcholine, alone or in mixture with lipids enriched along with the protein within lipid rafts: cholesterol, sphingomyelin, and GM1 ganglioside. The highest V max was recorded for a phosphatidylcholine/10% GM1 mixture (143 +/- 5 nmol of substrate hydrolyzed per minute per microgram of protein), while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes (M:M:M) (57 +/- 3 and 52 +/- 3, respectively). No significant differences in K m were detected. The protein segregation, assessed using the chemical cross-linker bis(sulfosuccinimidyl)suberate, increased with the protein:lipid ratio, within the 1:1200-1:4800 protein:lipid molar ratio range, but did not affect enzyme activity. The activity decreased when the order of the lipid bilayers was increased, higher for those containing cholesterol, as judged by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Finally, the GPI-enzyme activity was affected by membrane curvature. This result was suggested by a strong inverse correlation (Pearson's correlation coefficient = 0.91; p < 0.0001) between activity and liposome diameter, measured by laser light scattering and ranging between 59 +/- 6 nm for a phosphatidylcholine/10% GM1 mixture (displaying the highest activity) and 188 +/- 25 nm for a phosphatidylcholine/30% cholesterol mixture and 185 +/- 23 nm for raft-mimicking liposomes (displaying the lowest activities). The activity-membrane curvature relationship was further confirmed by comparing the activity of proteoliposomes having different sizes but identical lipid compositions. These data open the possibility that the activity of GPI-anchored enzymes may be modulated by membrane microenvironment features, in particular by membrane curvature and cholesterol-enriched ordered microenvironments, such as those of lipid rafts.     

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.     

Interaction of the toxic plant protein--ricin, with model membranes. A fluorescence method of study]

The fluorescence method has been used to investigate ricin and its isolated subunits interaction with some model membranes. Three liposome types were used as a model of biological membrane: 1) liposomes constructed from lecithin and cholesterol (9:1, M:M) 2) from ganglioside receptors GM1 and 3) from the mixture of GM1, lecithin and cholesterol (1:9:1). Interaction of the protein with liposome evokes changes in the parameters of both intrinsic protein fluorescence and fluorescence of the covalently bound dansyl. Binding constants were calculated from a decrease of the intrinsic fluorescence intensity as well as from the changes in the dansyl rotation anisotropy. Measurements were carried out at neutral and acidic pH. There was good correlation of the results obtained by different methods. It was shown that association constants were different for intact ricin and its subunits. The constants also depend on liposome composition and pH of the solution. The present study has demonstrated that interaction of ricin with liposome is accounted for not only by receptor centers but also by other hydrophobic regions of ricin that are inaccessible in the native toxin and may represent the region of the subunits interaction.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Modification of the lipid composition of neuroblastoma C1300 cells using liposomes

An essential increase in the total cholesterol content with a significant growth in the level of its esters was observed in neuroblastoma C1300 cells differentiated by means of 5'-bromodesoxyuridine and incubated for 1 h with lecithin-cholesterol liposomes (1:1 mol/mol). These cells also displayed an increased amount of polyunsaturated fatty acids in the composition of lecithin molecules and the appearance of lysolicethin as well. Incubation of the cells with lecithin liposomes was accompanied by a decrease in the content of cholincholesterol in the cells. In this case the level of cholesterol esters decreased to a greater extent than the level of free cholesterol. In lecithin molecules of these cells there occurred a relative increase of saturated fatty acids. The cells can retain viability and compensate changes caused by cholesterol excess during their incubation with lecithin-cholesterol liposome for 60-90 min. A longer incubation with liposomes results in a sharp drop of cell viability.     

Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane

When the liposome membrane is exposed to the alpha-toxin of Staphylococcus aureus, fluorescence of the tryptophan residue(s) of the toxin molecule increases concomitantly with the degree of toxin-hexamer formation (Ikigai, H., and Nakae, T. (1985) Biochem. Biophys. Res. Commun. 130, 175-181). In the present study, the toxin-membrane interaction was distinguished from the hexamer formation by the fluorescence energy transfer from the tryptophan residue(s) of the toxin molecule to the dansylated phosphatidylethanolamine in phosphatidylcholine liposome. Measurement of these two parameters yielded the following results. The effect of the toxin concentration and phospholipid concentration on these two parameters showed first order kinetics. The effect of liposome size on the energy transfer and the fluorescence increment of the tryptophan residue(s) was only detectable in small liposomes. Under moderately acidic or basic conditions, the fluorescence energy transfer always preceded the fluorescence increment of the tryptophan residue(s). The fluorescence increment at 336 nm at temperatures below 20 degrees C showed a latent period, whereas the fluorescence energy transfer did not. These results were thought to indicate that when alpha-toxin damages the target membrane, the molecule interacts with the membrane first, and then undergoes oligomerization within the membrane.     

Interaction of influenza hemagglutinin amino-terminal peptide with phospholipid vesicles: a fluorescence study

We have studied tryptophan fluorescence from a 20-residue synthetic peptide corresponding to the amino terminal of the HA2 subunit of the influenza virus hemagglutinin protein, a putative "fusion" peptide. Decay-associated spectra have been obtained at pH 7.4 and at pH 5 (the optimal pH for influenza virus fusion) in the presence and absence of liposomes. We demonstrate that a blue shift in the total steady-state fluorescence spectrum upon binding to liposomes is due to a movement in characteristic emission wavelength and increased lifetime of one of the resolved spectral components. In contrast, a further shift after lowering the pH is the product of a redistribution in the relative amplitudes of spectral components. Also, each decay component is quenched by spin-labels or anthroxyl groups normally located within the hydrocarbon interior of the membranes. Calculations are presented leading to an estimate of the distance of the tryptophan residue from the bilayer center, suggesting that the tryptophan residues are at or near the hydrocarbon-polar interface. No gross positional change was detected between pH values. Rotational depolarization is shown to be retarded by liposome binding, more so at low pH.     

Serum-induced leakage of liposome contents

Efflux of contents from small unilamellar vesicles of various compositions, containing a highly quenched fluorescent compound (calcein, 175 mM) was determined as a function of temperature in the presence and absence of human serum. Efflux of calcein from the liposomes was monitored as an increase in fluorescence as calcein became dequenched upon release from the liposomes. The presence of serum significantly increased liposome leakage in all cases. Incorporation of increasing molar ratios of cholesterol into liposomes reduced leakage of calcein from liposomes incubated with buffer and with serum. Leakage was significantly faster from liposomes with an osmotic gradient across the membrane (higher inside) than from equiosmolar liposomes. The leakage of [¹⁴C]sucrose from egg lecithin liposomes at 37°C was also dramatically increased in the presence of serum.