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.

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.

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.

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.

Molecular interactions between sphingomyelin and phosphatidylcholine in phospholipid vesicles.

The molecular interactions between DPPC and sphingomyelin have been examined using differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). It has been observed using DSC that the mixtures of DPPC and sphingomyelin show a single peak, which indicates that they are very miscible. It has been shown, by means of FT-IR, that the incorporation of sphingomyelin into DPPC does not modify the gauche/all-trans ratio at temperatures either above or below that of the phase transition. However, the librational motion of the fatty acyl chains was affected, although only above the phase transition. Mixing sphingomyelin and DPPC induced a conformational change in the polar region of DPPC, as deduced from the changes observed in the frequencies of the sn-1 and sn-2 C = O groups of DPPC with relation to pure DPPC and also in the polar region of sphingomyelin as deduced from the change in the frequency of the amide band. The phosphate group of sphingomyelin was observed to participate in hydrogen bonds between the molecules of sphingomyelin and, possibly, DPPC. Some possibilities of the interaction between the molecules of DPPC and sphingomyelin are discussed.

Effects of platelet-activating factor and related lipids on dielaidoylphosphatidylethanolamine by DSC, FTIR and NMR.

The effect of platelet-activating factor (1-O-hexadecyl-2-acetyl-sn- glycero-3-phosphocholine, PAF) and two related molecules, 1-O-hexadecyl-sn-glycero-3-phosphocholine (LPAF) and 1-palmitoyl-sn-glycero-3- phosphocholine (LPC) on dielaidoylphosphatidylethanolamine (DEPE) lipid structure and polymorphism has been studied by differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) and 31P nuclear magnetic resonance (31P-NMR) spectroscopies. From the interaction of these molecules with DEPE it is concluded that all of them stabilize the lamellar phase with respect to the hexagonal HII phase and this effect is clear even at concentrations of these compounds as low as 1 mol%. It is also shown that, although they perturb the gel to liquid-crystalline phase transition of DEPE up to a similar extent, fluidizing the membrane, PAF but not LPAF or LPC, induces the presence of more than one peak in the calorimetric profile. Moreover, FTIR data indicate that lateral phase separations formed by PAF-rich phases are taking place. Remarkably, delta H of the main transition decreases at concentrations lower than 2 mol% but remains nearly constant up to 30 mol%. 31P-NMR measurements showed that all these molecules were capable of inducing isotropic signals in the spectra produced by molecules associated to membranes before micellization of the vesicles.

The interaction of vitamin K1 with phospholipid vesicles.

Vitamin K1 is a component of the electron transport chain in chloroplasts and also an activator of carboxylases present in microsomes of different tissues. In order to understand its mechanism of action it is necessary to know its interactions, localization and organization in the phospholipid bilayer. We have studied this question using reconstituted systems of vitamin K1 incorporated in DPPC multibilayer vesicles, using DSC, DPH anisotropy and FT-IR spectroscopy. DSC shows that the pretransition is modified and disappears as the concentration of vitamin K1 in the bilayer increases. The main transition is also affected, with a decrease of Tm and delta H with increasing concentrations of vitamin K1. Furthermore, a second peak appears at high concentrations of vitamin K1, which is indicative of a lateral phase separation of a phase rich in vitamin K1. Fluorescence measurements using DPH show, in agreement with the calorimetric measurements, that the phase transition is shifted to lower temperatures and the anisotropy is increased above Tm but, interestingly, not below Tm. FT-IR spectroscopic measurements are also in good agreement with the calorimetric and fluorescence results, indicating that vitamin K1 induced a broadening and a shift to lower temperatures in the phase transition. It is deduced from the variation of the frequency parameter of the CH2 stretching vibration band with temperature that vitamin K1 perturbs the average number of gauche and all-trans conformers of DPPC, only during the phase transition interval but neither at temperatures above nor below the phase transition. However, the bandwidth parameter of this vibration indicated a perturbation above, but not below, the phase transition. The possible relationship between these observations and those coming from fluorescence depolarization of DPH are discussed. Finally, it is concluded from the observation of the C=O stretching mode that vitamin K1 does not produce a very strong perturbation of the interfacial region of the membrane of the type given by, for example, cholesterol.

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.

Diffusivity and structural polymorphism in some model stratum corneum lipid systems.

Mixtures of model stratum corneum lipids were prepared in water from cholesterol, six fatty acids and ceramides. The influence of composition on the polymorphism of these mixtures and also on the diffusivity of a model drug within them, Dlip, was determined. The former was obtained from X-ray diffraction and Fourier transform infrared spectrometry, and the latter from a diffusional release model. An L beta structure was formed for the composition approximating that of the extracellular lipids in intact human abdominal stratum corneum. Dlip was independent of water content in the range 20-40% w/w, with the bilayers showing one dimensional swelling without lateral expansion. Although removal of the ceramides did not result in a significant alteration in Dlip, crystalline cholesterol now appeared. The ceramides were, therefore, necessary for solubilization within the fatty acid bilayers of the large proportion of cholesterol present in the lipid fraction of intact SC. They were also responsible for a thermal L alpha-HII transition observed at approx. 68 degrees. At the concentration in which it exists in intact SC, cholesterol also had only a minimal effect on Dlip, but was necessary to suppress HII phase formation within the fatty acids and ensure an L beta structure. All lipid mixtures that had an L beta structure presented a diffusional barrier approx. 1 order of magnitude greater than that of an unstructured, isotropic lipid mixture. HII structures formed at cholesterol/fatty acid proportions less than approx 8:92 mol% and appeared more permeable than L beta ones. All the results indicate that the diffusional barrier within the model lipid mixtures is guaranteed essentially by the presence of an L beta phase. Although the ceramides and cholesterol exert no intrinsic influence on the magnitude of Dlip, their presence in necessary for the existence of an L beta phase at 33 degrees that is free of both crystalline cholesterol and HII character.

Interaction of sphingosine and stearylamine with phosphatidylserine as studied by DSC and NMR.

The interaction of sphingosine (SP) and stearylamine (SA) with dipalmitoylphosphatidylserine (DPPS) has been studied by using differential scanning calorimetry (DSC) and phosphorus nuclear magnetic resonance (31P-NMR). DSC showed that SP and SA rigidified the membranes, forming an azeotropic mixture with DPPS. The azeotropic mixture which was formed between DPPS and SP was found at a DPPS/SP molar ratio of 2:1 whereas SA and DPPS formed an azeotropic mixture at a DPPS/SA molar ratio of 1:1. An eutectic point was observed at 85 mol% of SP and 90 mol% of SA in DPPS. 31P-NMR showed the presence of a lamellar phase at DPPS/SP and DPPS/SA molar ratios lower than 1:1, whereas at higher molar ratios and at high temperatures, besides the lamellar phase, an isotropic component was detected. It was found that, at physiological pH, both SP and SA were protonated in a large extent, i.e., positively charged, since their apparent pK in the membrane were 9.1 and 8.9, respectively. The results reported in this work may be relevant to understand a number of biological effects produced by these positively charged molecules, due to their electrostatic interaction with negatively charged phospholipids.

Influence of vitamin E on phosphatidylethanolamine lipid polymorphism.

The effect of vitamin E, in its major form alpha-tocopherol and its synthetic analog alpha-tocopheryl acetate, on phosphatidylethanolamine lipid polymorphism has been studied by mean of differential scanning calorimetry and 31P-nuclear magnetic resonance techniques. From the interaction of these tocopherols with dielaidoylphosphatidylethanolamine it is concluded that both molecules promote the formation of the hexagonal HII phase at temperatures lower than those of the pure phospholipid. When the tocopherols were incorporated in the saturated dimiristoylphosphatidylethanolamine, which has been shown not to undergo bilayer to hexagonal HII phase transition, up to 90 degrees C, they induce the phospholipid to partially organize in hexagonal HII phase. From our experiments it is shown that alpha-tocopherol is more effective than its analog in promoting HII phase in these systems. It is also shown that, while alpha-tocopheryl acetate does not significantly perturb the gel to liquid-crystalline phase transition of dimirystoylphosphatidylethanolamine, alpha-tocopherol does so and more than one peak appears in the calorimetric profile, indicating that lateral phase separations are taking place.

Fourier transform infrared spectroscopic studies on the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum.

Fourier transform infrared spectroscopy has been applied to the study of the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum. An attempt is made to quantitatively assess the various secondary structures present. Values of 45% alpha-helix, 32% beta-sheet and 23% turns were obtained. A comparison is made of these results and those obtained using other techniques such as CD and Raman spectroscopy. The various assumptions inherent in the present procedure are discussed. The effect of various ligands, e.g. Ca2+, vanadate, ATP and phosphate, upon the structure were investigated. Upon binding these ligands no marked spectral changes were observed.

A phase behavior study of mixtures of sphingosine with zwitterionic phospholipids.

The interactions of sphingosine (SPH) with dipalmitoylphosphatidylcholine (DPPC) and dielaidoylphosphatidylethanolamine (DEPE) have been studied by means of differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (31P-NMR). Experiments were carried out with the fully protonated form of SPH, at pH 6.0. DSC studies showed that the main Tc transition temperature of DPPC was perturbed by the presence of SPH so that Tc of the mixture was higher than those of pure components at concentrations of SPH up to 50 mol%, with an azeotropic point at 30 mol% of SPH. At higher concentrations solid phase separations were observed from 70 to 95 mol% of SPH with an eutectic point at 90 mol% of SPH. 31P-NMR showed lamellar phases in DPPC/SPH mixtures, at all the range of concentrations. The behavior of DEPE/SPH mixtures was somewhat different since no azeotropic point was detected, the gel to liquid-crystalline transition being depressed by the presence of SPH, and an eutectic point was detected at 60 mol%. Solid phase immiscibilities were present between 50 mol% and 85 mol% of SPH. It is also remarkable that the liquid-crystalline to hexagonal HII phase transition of DEPE was only slightly shifted to lower temperatures at concentrations of SPH lower than 33 mol% of SPH but, this transition disappeared at concentrations of SPH higher than 33 mol% of SPH, so that isotropic phases were formed instead, as seen through 31P-NMR. The present results show the importance of taking into account the effects appearing in mixtures of SPH with zwitterionic phospholipids when considering their influence on the organization of biomembranes.

Influence of retinoids on phosphatidylethanolamine lipid polymorphism.

The interaction of all-trans-retinoic acid and all-trans-retinol with dielaidoylphosphatidylethanolamine has been studied by differential scanning calorimetry and 31P-NMR spectroscopy. Increasing concentrations of all-trans-retinoic acid up to a mol fraction of 0.09 were found to induce shifts to lower temperatures of both the L beta to L alpha and L alpha to hexagonal-HII phase transitions, with a slight decrease in the enthalpy change of the transitions. At higher concentrations no further effects on the transitions were observed, and this is interpreted as indicative of a limited miscibility of retinoic acid with the phospholipid. 31P-NMR spectroscopy confirmed that the L alpha to hexagonal-HII phase transition was shifted to lower temperatures in the presence of retinoic acid. On the other hand increasing concentrations of all-trans-retinol up to a mol fraction of 0.166, induced a progressive shift of the L beta to L alpha and the L alpha to hexagonal-HII phase transitions to lower temperatures. At higher concentrations the main gel to liquid-crystalline phase transition was further displaced to lower temperatures and the lamellar to hexagonal-HII phase transition was not observed in the thermograms. 31P-NMR spectroscopy indicated that retinol was able of inducing the phospholipid to adopt the hexagonal-HII phase at temperatures even below the main gel to liquid-crystalline phase transition temperature of the pure phospholipid.

Infrared spectroscopic study of the interaction of diacylglycerol with phosphatidylserine in the presence of calcium.

The interaction of 1,2-dipalmitoylglycerol (DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in aqueous dispersion in the presence and in the absence of Ca2+ by using Fourier transform infrared spectroscopy (FT-IR) and 45Ca(2+)-binding. FT-IR showed that DPG increased the phase transition of DPPS and induced a rigidification of the DPPS/DPG-Ca2+ complex. In the absence of Ca2+, the incorporation of DPG produced an increase in the proportion of dehydrated carbonyl groups in the mixture of DPPS plus DPG whereas, in the presence of Ca2+, DPG suppressed the solid-solid phase transition of phosphatidylserine-Ca2+ complexes. The phosphate band of DPPS was analyzed using a multivariate statistical analysis, indicating that DPG induced a higher dehydration of the PO2- group in the presence of subsaturating Ca2+ concentrations. Even very low concentrations of DPG, such as 2 mol%, already produced a significant effect. In the presence of both DPG and Ca2+, dehydration of DPPS increased, so that full dehydration was reached at a DPPS/Ca2+ molar ratio of 2.94 instead of 2.04 as observed for pure DPPS. However, the stoichiometry of the binding of Ca2+ to DPPS was not significantly altered by the inclusion of DPG as revealed by 45Ca(2+)-binding experiments, indicating that, in this situation, full dehydration of the PO2- groups of DPPS was reached when approx. 2 out of every 3 molecules of DPPS were binding Ca2+. The effects reported here for the interaction of DPG with DPPS may be significant for a number of biological situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes or the activation of protein kinase C, where the dehydration effect produced by diacylglycerols may explain, at least in part, their effects.

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.

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.

Diacylglycerol, phosphatidylserine and Ca2+: a phase behavior study.

The interaction of 1,2-dipalmitoylglycerol (1,2-DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in the presence and in the absence of Ca2+ by using differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (31P-NMR). In the absence of Ca2+, DSC showed that 1,2-DPG increased the phase transition of DPPS, effect already noticed at very low 1,2-DPG concentrations, whereas lipid immiscibilities were detected at concentrations of 1,2-DPG higher than about 30 mol%. 31P-NMR indicated that lamellar phases were always present at concentration of 1,2-DPG lower than about 35 mol%, but at higher concentrations non-lamellar phases may be present in the fluid phase. As observed by DSC, the apparent pKa of the carboxyl group of DPPS was increased slightly in the presence of 1,2-DPG. In the presence of Ca2+, the effect of 1,2-DPG was to further increase the temperature of the onset of the phase transition, indicating an stabilization of the most rigid phase in the DPPS/1,2-DPG/Ca2+ samples. Even concentrations of 1,2-DPG as low as 1 mol% of the total lipid already produced a noticeable effect. Moreover, lipid immiscibilities were apparent at concentrations of 1,2-DPG higher than 20 mol%. Furthermore, the transition of the DPPS/Ca2+ complex observed by DSC at 155 degrees C was perturbed by the presence of 1,2-DPG, indicating a change in the structure of the crystalline complex. Interestingly, the effect of non-saturating Ca2+ concentrations on the DPPS phase transition was enhanced by the presence of 1,2-DPG. The effect reported here may be significant for a number of situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes, where diacylglycerol may facilitate Ca(2+)-induced fusion, or the activation of enzymes such as protein kinase C and phospholipases.

Capsaicin affects the structure and phase organization of phospholipid membranes.

Capsaicin is a natural compound with pharmacological and toxicological effects, which given its hydrophobicity, can influence the structure of membranes. The interaction of capsaicin with model membranes of dipalmitoylphosphatidylcholine and dielaidoylphosphatidylethanolamine has been studied by using differential scanning calorimetry, fluorescent probe spectroscopy and 31P-nuclear magnetic resonance. Capsaicin remarkably affects the phase transition of dipalmitoylphosphatidylcholine, shifting the transition temperature to lower values, and giving rise, at relatively high capsaicin concentrations, to the appearance of two peaks in the thermogram. These peaks may correspond to separated phases as indicated by the partial phase diagram. Whereas capsaicin did not affect the fluorescence polarization of the probes diphenylhexatriene and trimethylammonium-diphenylhexatriene, it clearly affected that of the probe 2-anthroyloxystearic acid, indicating that the perturbation produced by capsaicin on the membrane would be mainly at the position where this fluorophore is located. On the other hand, capsaicin, at relatively low concentrations, gives rise to immiscible phases in the presence of dielaidoylphosphatidylethanolamine and decrease the temperature of the lamellar to hexagonal HII phase transition. At concentrations of capsaicin higher than 0.3 mol fraction, isotropic phases were detected. The possible implications of the effects of capsaicin on biological membranes are discussed.