Secondary structure determination by FTIR of an archaeal ubiquitin-like polypeptide from Natrialba magadii.

The ubiquitin protein belongs to the ß-grasp fold family, characterized by four or five ß-sheets with a single α-helical middle region. Ubiquitin-like proteins (Ubls) are structural homologues with low sequence identity to ubiquitin and are widespread among both eukaryotes and prokaryotes. We previously demonstrated by bioinformatics that P400, a polypeptide from the haloalkaliphilic archaeon Natrialba magadii, has structural homology with both ubiquitin and Ubls. This work examines the secondary structure of P400 by Fourier transform infrared spectroscopy (FTIR). After expression in Escherichia coli, recombinant P400 (rP400) was separated by PAGE and eluted pure from zinc-imidazole reversely stained gels. The requirement of high salt concentration of this polypeptide to be folded was corroborated by intrinsic fluorescence spectrum. Our results show that fluorescence spectra of rP400 in 1.5 M KCl buffer shifts and decreases after thermal denaturation as well as after chemical treatment. rP400 was lyophilized and rehydrated in buffer containing 1.5 M KCl before both immunochemical and FTIR tests were performed. It was found that rP400 reacts with anti-ubiquitin antibody after rehydration in the presence of high salt concentrations. On the other hand, like ubiquitin and Ubls, the amide I' band for rP400 shows 10% more of its sequence to be involved in ß-sheet structures than in α-helix. These findings suggest that P400 is a structural homologue of the ubiquitin family proteins.

Membrane fluidity determines sensitivity of filamentous fungi to chitosan.

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan-resistant but not chitosan-sensitive fungi. The plasma membranes of chitosan-sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan-resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady-state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.

Hepatitis C virus core protein binding to lipid membranes: the role of domains 1 and 2.

We have analysed and identified different membrane-active regions of the Hepatitis C virus (HCV) core protein by observing the effect of 18-mer core-derived peptide libraries from two HCV strains on the integrity of different membrane model systems. In addition, we have studied the secondary structure of specific membrane-interacting peptides from the HCV core protein, both in aqueous solution and in the presence of model membrane systems. Our results show that the HCV core protein region comprising the C-terminus of domain 1 and the N-terminus of domain 2 seems to be the most active in membrane interaction, although a role in protein-protein interaction cannot be excluded. Significantly, the secondary structure of nearly all the assayed peptides changes in the presence of model membranes. These sequences most probably play a relevant part in the biological action of HCV in lipid interaction. Furthermore, these membranotropic regions could be envisaged as new possible targets, as inhibition of its interaction with the membrane could potentially lead to new vaccine strategies.

The cytotoxic properties of a plant lipid transfer protein involve membrane permeabilization of target cells.

AIMS: To determine whether Ha-AP10, a member of the plant lipid transfer proteins (LTPs) family produces a direct cytotoxic effect on fungal cells mediated by membrane permeabilization. LTPs can inhibit fungal growth and are considered members of the ubiquitous class of antimicrobial peptides. However, the way they exert their effects on target cells is not yet understood. METHODS AND RESULTS: Viability assays demonstrate that Ha-AP10 acts as a fungicidal compound but no harmful effect is observed on plant cells. Liposome leakage assays show that the protein induces a moderate release of fluorescent probes encapsulated in model membranes, indicating its ability to interact with phospholipids. Using a fluorescent indicator of damage at the membrane level, we demonstrate that Ha-AP10 is able to induce the permeabilization of intact fungal spores in a dose-dependent manner. CONCLUSION: The results presented here demonstrate the permeabilization of fungal spores caused by Ha-AP10. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first demonstration of fungal membrane damage by an LTP, giving a clue to elucidate the basis of its antimicrobial properties.

Membranotropic effects of the antibacterial agent Triclosan.

Triclosan is a broad-spectrum hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. To gain further insight into the mode of action of Triclosan we examined its effects on membranes by performing leakage titrations of different oral bacteria and studying its interaction with model membranes through the use of different biophysical techniques. There was negligible efflux of intracellular material from Streptococcus sobrinus at the minimal inhibitory concentration of Triclosan; whatever leakage did occur commenced only at much higher concentrations. In contrast, no leakage was observed at even the minimal bactericidal concentration for Porphyromonas gingivalis. Triclosan decreased the onset temperature of the gel to liquid-crystalline phase transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-3-[phospho-rac-glycerol] membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Steady-state fluorescence anisotropy measurements of different phospholipid/Triclosan samples using 3-(p-6-phenyl-1,3,5-hexatrienyl)-phenylpropionic acid were consistent with the calorimetric data. Incorporation of increasing amounts of Triclosan into 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) vesicles induced the nonlamellar H(II) hexagonal phase at low temperatures and new immiscible phases at temperatures below the main transition of DEPE. Taking these results together suggests that the antibacterial effects of Triclosan are mediated at least in part through its membranotropic effects, resulting in destabilized structures which compromise the functional integrity of cell membranes without inducing cell lysis.

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes.

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.

Interaction of alpha-melanocyte stimulating hormone with binary phospholipid membranes: structural changes and relevance of phase behavior.

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.

Structure and interaction with membrane model systems of a peptide derived from the major epitope region of HIV protein gp41: implications on viral fusion mechanism.

The HIV-1 gp41 envelope protein mediates entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into viral fusion mechanisms, we have investigated by infrared, fluorescence, and nuclear magnetic resonance spectroscopies and calorimetry a fragment of 19 amino acids corresponding to the immunodominant region of the gp41 ectodomain, a highly conserved sequence and major epitope. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds and interacts with negatively charged phospholipids, changes its conformation in the presence of a membraneous medium, and induces leakage of vesicle contents as well as a new phospholipid phase. These characteristics might be important for the formation of the fusion-active gp41 core structure, promoting the close apposition of the two viral and target-cell membranes and therefore provoking fusion.

A fluorescence study of the interaction and location of (+)-totarol, a diterpenoid bioactive molecule, in model membranes.

(+)-Totarol, a diterpene extracted from Podocarpus totara, has been reported as a potent antioxidant and antibacterial agent. Although the molecular mechanism of action of this hydrophobic molecule remains unknown, recent work made in our laboratory strongly suggests that it could be lipid-mediated. Since (+)-totarol contains a phenolic ring, we have studied the intrinsic fluorescent properties of this molecule, i.e., quantum yield, lifetime, steady-state anisotropy and emission spectra, both in aqueous and in phospholipid phases, in order to obtain information on the interaction and location of (+)-totarol in biomembrane model systems. The phospholipid/water partition coefficient of (+)-totarol was found to be very high (K(p)=1.8x10(4)), suggesting that it incorporates very efficiently into membranes. In order to estimate the transverse location (degree of penetration) of the molecule in the fluid phase of DMPC model membranes, the spin labelled fatty acids 5-NS and 16-NS were used in differential quenching experiments. The results obtained show that (+)-totarol is located in the inner region of the membrane, far away from the phospholipid/water interface. Since (+)-totarol protects against oxidative stress, its interaction with an unsaturated fatty acid, trans-parinaric acid, was studied using fluorescence resonance energy transfer. No significant interactions were observed, molecules of trans-parinaric acid distributing themselves randomly amongst those of (+)-totarol in the phospholipid membrane.

Structure of the Alzheimer beta-amyloid peptide (25-35) and its interaction with negatively charged phospholipid vesicles.

The secondary structure of amyloid betaAP(25-35) peptide was studied in pure form and in the presence of different phospholipid vesicles, by using Fourier transform infrared spectroscopy (FT-IR). Pure peptide aggregated with time, forming fibrils with beta-structure. Phospholipid vesicles formed by negatively charged phospholipids such as 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (Myr2PtdSer), 1,2-dimyristoyl-sn-glycerol-3-phospho-rac-1-glycerol (Myr2PtdGro) and 1,2-dimyristoyl-sn-glycerol 3-phosphate (Myr2PtdH), greatly accelerated the aggregation of the peptide. However, the presence of vesicles formed by the zwitterionic phospholipid, 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine (Myr2PtdCho), slowed down the aggregation process. Differential scanning calorimetry (DSC) measurements showed that the effect of betaAP(25-35) on the gel to crystal liquid phase transition was small at neutral pH for negatively charged phospholipids and practically nil for Myr2PtdCho. In the case of Myr2PtdSer the effect was also zero at pH 9 but the effect was large at pH 3. The effect on Myr2PtdH was not, however, very dependent on pH. These results were fully confirmed by the observation through FT-IR of the change with temperature of the CH2 antisymmetric stretching vibration. The case of Myr2PtdGro was special as this phospholipid presents polymorphism giving solid quasicrystalline phases when it is not sufficiently hydrated, and it is remarkable that betaAP(25-35) was able to induce the formation of crystalline phases in samples prepared through a method which ensure a good hydration of phospholipid. These results show that the interaction of amyloid betaAP(25-35) peptide with phospholipids is based on electrostatic interactions, that these interactions favour the aggregation of the peptides, and that the presence of the aggregates may disturb the lipid-water interphase of the membrane.

Correlation between protein kinase C alpha activity and membrane phase behavior.

Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.

The phase behavior of aqueous dispersions of unsaturated mixtures of diacylglycerols and phospholipids.

The phase behavior of mixtures of 1-palmitoyl-2-oleoyl-sn-glycerol (1,2-POG) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was studied by using DSC, small-angle X-ray diffraction and 31P-NMR. The results have been used to construct phase diagrams for both type of mixtures, in the 0-45 degreesC range. It is concluded that 1, 2-POG form complexes in the gel phases with both POPC and POPS. In the case of POPC, two complexes are postulated, the first one at a 1, 2-POG/POPC molar ratio of 40:60, and the second one at 70:30, defining three different regions in the phase diagram. Two eutectic points are proposed to occur: one at a very low 1,2-POG concentration and the other at a 1,2-POG concentration slightly lower than 70%. In the case of the 1,2-POG/POPS mixtures, the pattern was similar, but the first complex was seen to happen at a higher concentration, about 50 mol% of 1,2-POG, whereas the second was found at 80 mol% of 1,2-POG. This indicated a bigger presence of 1,2-POG in the complexes with POPS than with POPC. In the first region of the phase diagram, i.e. at concentrations of 1,2-POG lower than that required for the formation of the first complex, and at temperatures above the phase transition, lamellar phases were seen in all the cases. In region 2 of the phase diagram, i.e. at concentrations where the first and the second complexes coexist, a mixture of lamellar and non-lamellar phases was observed. Finally, at high concentrations of 1,2-POG, non-lamellar phases were detected as predominant, these phases being of an isotropic nature, according to 31P-NMR. An important conclusion of this study is that, using unsaturated lipids, similar to those found in biological membranes, it has been shown that diacylglycerols are found separated in domains, and that this process starts at very low concentrations of diacylglycerols. The formation of separated domains enriched in diacylglycerol is biologically relevant as it will allow them to have important effects on the membrane structure besides the fact that their concentration in the biomembrane is relatively low.

The use of FT-IR for quantitative studies of the apparent pKa of lipid carboxyl groups and the dehydration degree of the phosphate group of phospholipids.

Fourier-transform infrared spectroscopy (FT-IR) has been applied to the quantitative study of the dehydration of the phosphatidylserine phosphate group in the presence of Ca2+ exerted by different molecules, such as diacylglycerol, sphingosine and stearylarnine, by using a partial least-squares statistical procedure. By using this method it was observed that diacylglycerol enhanced the dehydration of this PO2- group produced by Ca2+ whereas the amino-bases sphingosine and stearylamine protected the phosphate group from the dehydration produced by Ca2+ due to the very strong electrostatic interaction established. The apparent pKa of lipid carboxyl groups can also be estimated by using FTIR. The method consisted in quantifying the absorbance intensities due to the protonated and the unprotonated forms of the specific group being studied. The pKa of the carboxyl group of [1-13C]-palmitic acid included in dipalmitoylphosphatidylcholine membranes was found to be 8.7, a value much higher than that estimated from a molecular solution of the fatty acid. It was observed using the same method that the pKa of free fatty acids in model stratum corneum lipid mixtures was in the range 6.2-7.3 increasing with the preponderance of oleic acid over palmitic acid. Finally the pKa of the carboxyl group of phosphatidylserine was shifted from 4.6 in the pure phospholipid to 2.1 and 2.2 in the presence of equimolar sphingosine and stearylamine, respectively, as a consequence of electrostatic interactions.

Location of the toxic molecule abietic acid in model membranes by MAS-NMR.

Abietic acid, the major component of conifer oleoresin, is an environmental toxic molecule with potential hazard to animal and plant life. Being amphipatic, the study of its location and the interaction with membrane components is important to get insight into the mechanism of its toxic action. High resolution magic angle spinning natural abundance 13C nuclear magnetic resonance studies have been undertaken in order to assess its location in egg yolk phosphatidylcholine multilamellar vesicles model membranes. 13C spin-lattice relaxation times in the presence of Gd3+, a paramagnetic agent, of both the phospholipid and abietic acid molecules have been measured in order to obtain information on molecular distances (see J. Villalaín, Eur. J. Biochem. 241 (1996) 586-593). The molecule of abietic acid is placed in the upper part of the palisade structure of the membrane, its carboxyl group is in close proximity to the phospholipid ester groups and it does not extend beyond the C4/C7 carbons of the phospholipid molecule.

The interaction of abietic acid with phospholipid membranes.

Abietic acid is a major component of the oleoresin synthesized by many conifers and constitutes a major class of environmental toxic compounds with potential health hazard to animal, including human, and plant life. Being an amphipathic molecule, the study of the influence of abietic acid on the structure of membranes would be important to get insight into the mechanism of toxic action of the molecule. The interaction of abietic acid with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dielaidoylphosphatidylethanolamine (DEPE) has been studied by differential scanning calorimetry and 31P-nuclear magnetic resonance spectroscopy. It has been found that abietic acid greatly affects the phase transition of DPPC, shifting the transition temperature to lower values, giving rise to the appearance of two peaks in the thermogram and to the presence of fluid immiscible phases. In a similar way, the phase transition of DEPE, in the presence of abietic acid, was shifted to lower temperatures, and two peaks appeared in the thermograms. The temperature of the lamellar to hexagonal H(II) phase transition was also decreased by the presence of abietic acid, but phase immiscibilities were not detected. The possible implications of these effects on the action of abietic acid on biological membranes are discussed.

Location of cholesterol in model membranes by magic-angle-sample-spinning NMR.

High-resolution magic-angle-sample-spinning 13C-NMR was applied to determine the specific location of cholesterol in non-perturbed multilamellar model membranes formed by egg yolk phosphatidylcholine. 13C spin-lattice relaxation times of both the phospholipid and cholesterol molecules were measured in the absence and in the presence of Gd3+, a paramagnetic agent, in order to obtain information on molecular distances. The effect of Gd3+ on the spin-lattice relaxation times of the lipid resonances has an explicit distance dependence, allowing it to be used to evaluate relative distances on a molecular scale. It has been found that cholesterol is placed in such a position that it is not readily exposed to the solvent: the hydrophobic steroid ring is oriented parallel to the membrane phospholipids, the hydroxyl group is in close vicinity to the phospholipid ester carbonyl groups and the isooctyl side chain is deeply buried in the center of the membrane. These data are consistent with an organization such that mixtures of cholesterol and phospholipids present a packing similar to that found in interdigitated lipid bilayer systems.

Structural studies of synthetic peptides dissected from the voltage-gated sodium channel.

Peptides representing transmembrane regions of the alpha-subunit of the voltage-gated sodium channel were synthesised and their structures analysed, using 1H NMR and CD, in trifluoroethanol and in dodecylphosphocholine micelles. Sequence analysis suggests that the channel has six regions, S1 to S6, predicted to span the membrane in four homologous domains, designated, I, II, III and IV. Presented here are studies of representatives examples of possible single spanning segments (IS2, IS4, IVS4) and a double spanning segment, IS34, composed of segments IS3 and IS4. In addition, we investigated ISlink56, the putative linker region between segments IS5 and IS6. All of the peptides were found to have predominantly alpha-helical structures in both solvent systems. There was some evidence for bending of the longer helices but there was no discernible evidence for well-defined tertiary structure.

Metastability of dimiristoylphosphatidylethanolamine as studied by FT-IR and the effect of alpha-tocopherol.

The metastability of dimiristoylphosphatidylethanolamine (DMPE) has been studied by means of Fourier transform infrared spectroscopy (FT-IR), both in the absence and in the presence of alpha-tocopherol. Two different methods of hydration were used to prepare the samples, poorly hydrated and well hydrated, and the results have been compared with anhydrous DMPE. Poorly hydrated DMPE gave place to a high-melting phase formed upon melting from gel to L alpha at approx. 49 degrees C, with a new transition to L alpha at approx. 55 degrees C. However, well hydrated DMPE incubated at 4 degrees C for 49 days gave place to a subgel phase which was transformed by heating into a L beta phase at about 40 degrees C and this into a L alpha phase after further heating at 52 degrees C. The subgel phase was more hydrated and less rigid than the high-melting phase. On the other hand, alpha-tocopherol, when included in poorly hydrated DMPE, stabilized a high-melting phase, which was transformed by heating, directly into a L alpha. However, when a sample of DMPE containing alpha-tocopherol was incubated for 49 days at 4 degrees C a dehydrated solid phase different from the subgel and the high-melting phases was formed.

Apparent pKa of the fatty acids within ordered mixtures of model human stratum corneum lipids.

PURPOSE: The apparent pKa of the fatty acids within hydrated (30% w/w) model human stratum corneum (SC) lipid mixtures should be measured. METHODS: The degree of ionisation of the fatty acids was calculated as a function of pH using Fourier transform infra-red spectroscopy. The relative intensity of the stretching bands of the unionized and ionized carboxylic groups was determined and fitted to the relevant expression for ionic equilibrium of a monoprotic acid. The pKa was then calculated for increasing proportion of unsaturated fatty acid in the lipid mixture. RESULTS: Values for pKa in the range 6.2-7.3 were found, increasing with greater proportion of oleic acid. These are some 1.5-3 pH units higher than the pKas of fatty acids in molecular solution. CONCLUSIONS: As there exists a pH-gradient across the SC, the degree of ionisation will also vary. In the innermost SC layers, a pH of 7 will produce 90% ionization of the fatty acids and head-group repulsion will be great. At the SC surface, the pH of 5 will cause almost minimal head-group repulsion, tending to increase crystallinity and promote a bilayer structure.

Effect of sphingosine and stearylamine on the interaction of phosphatidylserine with calcium. A study using DSC, FT-IR and 45Ca(2+)-binding.

The lamellar gel to lamellar liquid-crystalline phase transition of dipalmitoylphosphatidylserine (DPPS) multilamellar membranes is abolished by the presence of Ca2+ at DPPS/Ca2+ molar ratios of 2:1 or lower. However, when equimolar sphingosine (SPH) or stearylamine (SA), which are positively charged at the pH studied in this work, were included in DPPS vesicles, the phase transition of DPPS was still observed by differential scanning calorimetry, even in the presence of very high Ca2+ concentrations such as a DPPS/Ca2+ molar ratio of 1:10. According to that, delta H was similar for samples formed by equimolar DPPS and SPH and SA, either in the presence or in the absence of Ca2+, whereas no phase transition was observed for the pure phospholipid in the presence of Ca2+ at molar ratios lower than DPPS/Ca2+ 2:1. 45Ca(2+)-binding experiments showed that for DPPS/SPH or DPPS/SA molar ratios of 2:1, only half of the Ca2+ was bound to DPPS with respect to pure DPPS, i.e., in the absence of SPH or SA. At concentrations of SPH or SA equimolar with DPPS, the Ca2+ binding was nearly abolished. The effect of SPH and SA on the the apparent pKapp of the carboxyl group of DPPS was also studied in the presence and in the absence of Ca2+ by using Fourier transform infrared spectroscopy. The dehydration of the phosphate group of DPPS induced by the binding of Ca2+ was followed through the observation of the PO2- antisymmetric stretching, and the percentage of dehydrated PO2- groups quantitatively assayed. It was again confirmed that, in the presence of equimolar concentrations of SPH or SA, Ca2+, at concentrations which are saturating for pure DPPS, was not bound at all to DPPS. It was also found that the pKapp was considerably shifted to lower values in the presence of the amino bases, decreasing from 4.6 in pure DPPS to 2.1 and 2.2 for the equimolar mixtures of DPPS with SPH and SA, respectively. These results show that SPH and SA, being positively charged molecules anchored in the membrane, are able of preventing the binding of positively charged ions such as Ca2+ through an electrostatic charge neutralization.