Search

Vitamin K1 and vitamin K2 play very important biological roles as members of chains of electron transport as antioxidants in membranes and as cofactors for the posttranslational modification of proteins that participate in a number of physiological functions such as coagulation. The interaction of these vitamins with dimyristoylphosphatidylcholine (DMPC) model membranes has been studied by using a biophysical approach. It was observed by using differential scanning calorimetry that both vitamins have a very limited miscibility with DMPC and they form domains rich in the vitamins at high concentrations. Experiments using X-ray diffraction also showed the formation of different phases as a consequence of the inclusion of either vitamin K at temperatures below the phase transition. However, in the fluid state, a homogeneous phase was detected, and a decrease in the thickness of the membrane was accompanied by an increase in the water layer thickness. 2H NMR spectroscopy showed that both vitamins K induced a decrease in the onset of the phase transition, which was bigger for vitamin K1, and both vitamins decreased the order of the membrane as seen through the first moment (M1). 1H NOESY MAS-NMR showed that protons located at the rings or at the beginning of the lateral chain of both vitamins K interacted with a clear preference with protons located in the polar part of DMPC. On the other hand, protons located on the lateral chain have a nearer proximity with the methyl end of the myristoyl chains of DMPC. In agreement with the 2H NMR, ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) indicated that both vitamins decreased the order parameters of DMPC. It was additionally deduced that the lateral chains of both vitamins were oriented almost in parallel to the myristoyl chains of the phospholipid.

Vitamin K

Idebenone is a synthetic analog of coenzyme Q; both share a quinone moiety but idebenone has a shorter lipophilic tail ending with a hydroxyl group. Differential scanning calorimetry experiments showed that both idebenone and idebenol widened and shifted the phase transition of 1,2-dipalmitoylphosphatidylcholine (DPPC) to a lower temperature and a phase separation with different concentrations of these molecules was observed. Also small angle X-ray diffraction and wide angle X-ray diffraction revealed that both, idebenone and idebenol, induced laterally separated phases in fluid membranes when included in DPPC membranes. Electronic profiles showed that both forms, idebenone and idebenol, reduced the thickness of the fluid membrane. (2)H NMR measurements showed that the order of the membrane decreased at all temperatures in the presence of idebenone or idebenol, the greatest disorder being observed in the segments of the acyl chains close to the lipid-water interface. (1)H NOESY MAS NMR spectra were obtained using 1-palmitoyl-2-oleoyl-phosphatidylcholine membranes and results pointed to a similar location in the membrane for both forms, with the benzoquinone or benzoquinol rings and their terminal hydroxyl group of the hydrophobic chain located near the lipid/water interface of the phospholipid bilayer and the terminal hydroxyl group of the hydrophobic chain of both compounds located at the lipid/water interface. Taken together, all these different locations might explain the different physiological behavior shown by the idebenone/idebenol compared with the ubiquinone-10/ubiquinol-10 pair in which both compounds are differently localized in the membrane.

Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.

α-Tocopherol is considered to carry on a very important role as antioxidant of membranes and lipoproteins and other biological roles as membrane stabilizers and bioactive lipids. Given its essential role, it is very important to fully understand its location in the membrane. In this work, the vertical location of vitamin E in saturated membranes has been studied using biophysical techniques. Small- and wide-angle X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both DMPC and DPPC, indicating its proximity to this surface. The quenching of the intrinsic fluorescence of α-tocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC. These results suggest that in both DMPC and DPPC membranes the chromanol ring is not far away from the surface of the membrane but within the bilayer. 1H-NMR-NOESY MAS-NMR studies showed that α-tocopherol is localized similarly in DMPC and DPPC membranes, with the chro...

α-Tocopherol is a natural preservative that prevents free radical chain oxidations in biomembranes. We have studied the location of α-tocopherol in model membranes formed by different unsaturated phosphatidylcholines, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC). Small angle X-ray diffraction revealed that α-tocopherol was well mixed with all the phospholipids. In all the cases only one lamellar phase was detected. Very modest changes occasioned by α-tocopherol were observed in the electron density profiles. The results obtained from quenching of α-tocopherol intrinsic fluorescence by acrylamide showed that this vitamin was inefficiently quenched in the four types of membranes, indicating that the fluorescent chromanol ring was poorly accessible for this hydrophilic quencher. Compatible with that, quenching by doxyl derivatives of phosphatidylcholines indicated that the chromanol ring was close in the four membranes to the nitroxide probe located at position 5. Quenching by doxyl-phosphatidylcholines also indicated that the efficiency of quenching was higher in POPC than in the other unsaturated phospholipids. 1H-MAS-NMR showed that α-tocopherol induced chemical shifts of protons from the phospholipids, especially of those bonded to carbons 2 and 3 of the acyl chains of the four phospholipids studied. The 1H-MAS-NMR NOESY results suggested that the lower part of the chromanol ring was located between the C3 of the fatty acyl chains and the centre of the hydrophobic monolayer for the four phospholipid membranes studied. Taken together, these results suggest that α-tocopherol is located, in all the membranes studied, with the chromanol ring within the hydrophobic palisade but not far away from the lipid–water interface.

The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide-phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the (31)P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T(1) relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T(1) values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. (13)C-MAS-NMR showed that T(1) values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T(1) affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.

The interaction between oxidized (ubiquinone-10) and reduced (ubiquinol-10) coenzyme Q 10 with dimyristoylphosphatidylcholine has been examined by differential scanning microcalorimetry, X-ray diffraction, infrared spectroscopy, and (2)H NMR. Microcalorimetry experiments showed that ubiquinol-10 perturbed considerably more the phase transition of the phospholipids than ubiquinone-10, both forms giving rise to a shoulder of the main transition peak at lower temperatures. Small angle X-ray diffraction showed an increase in d-spacing suggesting a thicker membrane in the presence of both ubiquinone-10 and ubiquinol-10, below the phase transition and a remarkable broadening of the peaks indicating a loss of the repetitive pattern of the lipid multilamellar vesicles. Infrared spectroscopy showed an increase in wavenumbers of the maximum of the CH 2 stretching vibration at temperatures below the phase transition, in the presence of ubiquinol-10, indicating an increase in the proportion of gauche isomers in the gel phase, whereas this effect was smaller for ubiquinone-10. A very small effect was observed at temperatures above the phase transition. (2)H NMR spectroscopy of perdeuterated DMPC showed only modest changes in the spectra of the phospholipids occasioned by the presence of coenzyme Q 10. These small changes were reflected, in the presence of ubiquinol-10, by a decrease in resolution indicating that the interaction between coenzyme Q and phospholipids changed the motion of the lipids. The change was also visible in the first spectral moment (M1), which is related with membrane order, which was slightly decreased at temperatures below the phase transition especially with ubiquinol-10. A slight decrease in M 1 values was also observed above the phase transition but only for ubiquinol-10. These results can be interpreted to indicate that most ubiquinone-10 molecules are localized in the center of the bilayer, but a considerable proportion of ubiquinol-10 molecules may span the bilayer interacting more extensively with the phospholipid acyl chains.

The C2 domain from protein kinase Cepsilon (PKCepsilon) binds to membranes but does not require Ca2+ to do so. This work examines the mode in which the conformation and organization of the phospholipids present in model membranes are altered by the presence of the C2 domain from PKCepsilon (C2-PKCepsilon). It is concluded from the results of differential scanning calorimetry that the protein shifted the temperature of the gel to the fluid phase transition of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), widening the transition and increasing it to a higher temperature. When POPA was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the changes in the transition were smaller and no phase separation was observed. Experiments performed using magic angle spinning NMR showed that this C2 domain specifically affected POPA when the phospholipid was mixed with POPC, as indicated by the downfield shift in the isotropic resonance of POPA, the widening of the resonance peak, the decrease in T2, and the decrease in T1 observed at all temperatures. All these effects were quite marked compared with the very small effect observed with POPC, indicating the specificity of the effect. The presence of the C2-PKCepsilon protein changed the conformation of the polar head group of POPA, as shown by infrared spectroscopy. All these results clearly illustrate the electrostatic interaction that takes place between this C2 domain and membranes which contain POPA in the absence of Ca2+.

Capsaicin is the chemical responsible for making some peppers spicy hot, but additionally it is used as a pharmaceutical to alleviate different pain conditions. Capsaicin binds to the vanilloid receptor TRPV1, which plays a role in coordinating chemical and physical painful stimuli. A number of reports have also shown that capsaicin inserts in membranes and its capacity to modify them may be part of its molecular mode of action, affecting the activity of other membrane proteins. We have used differential scanning calorimetry, X-ray diffraction, 31P NMR, and 2H NMR spectroscopy to show that capsaicin increases the fluidity and disorder of 1,2-palmitoyl-sn-glycero-3-phosphocholine membrane models. By using 1H NOESY MAS NMR based on proton-proton cross-peaks between capsaicin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine resonances, we determined the location profile of this molecule in a fluid membrane concluding that it occupies the upper part of the phospholipid monolayer, between the lipid-water interface and the double bond of the acyl chain in position sn-2. This location explains the disorganization of the membrane of both the lipid-water interface and the hydrophobic palisade. © 2015 American Chemical Society.

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.

The antineoplastic ether phospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phophocholine (ET-18-OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro-PCho)/dimyristoylglycerophosphoserine (Myr2Gro-PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET-18-OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET-18-OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET-18-OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel-to-fluid phase transition of this sample. On the other hand, protein kinase Cα (PKCα) activity progressively decreased when ET-18-OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone-shaped molecule. At higher concentrations, however, ET-18-OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET-18-OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET-18-OCH3 concentrations.

Resveratrol is a phytoalexin found in grapes and other foods that cancer chemopreventive and other biological activities have been attributed recently. We report that resveratrol is able to incorporate itself into model membranes in a location that is inaccessible to the fluorescence quencher, acrylamide. Differential scanning calorimetry revealed that resveratrol considerably affected the gel to liquid-crystalline phase transition of multilamellar vesicles made of phosphatidylcholine/phosphatidylserine and increased the temperature at which the fluid lamellar to H(II) inverted hexagonal transition took place in multilamellar vesicles made of 1,2-dielaidoyl-sn-phosphatidylethanolamine. Such a transition totally disappeared at 2.5 mM of resveratrol (resveratrol/lipid molar ratio of 2:1). This effect on 1, 2-dielaidoyl-sn-phosphatidylethanolamine polymorphism was confirmed through (31)P-NMR, which showed that an isotropic peak appeared at high temperature instead of the H(II)-characteristic peak of 42 mM of resveratrol (resveratrol/lipid molar ratio of 1.5:1). Finally, resveratrol inhibited PKCalpha when activated by phosphatidylcholine/phosphatidylserine vesicles with an IC(50) of 30 microM, whereas when the enzyme was activated by Triton X-100 micelles the IC(50) was 300 microM. These results indicate that the inhibition of PKCalpha by resveratrol can be mediated, at least partially, by membrane effects exerted near the lipid-water interface.

The capacity of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3) to modulate the polymorphic properties of dielaidoylphosphatidylethanolamine has been studied using biophysical techniques. Differential scanning calorimetry showed that ET-18-OCH3 depresses the onset of the Lbeta to Lalpha phase transition, decreasing also DeltaH of the transition. At the same time, the onset of the transition from Lalpha to inverted hexagonal HII phase was gradually increased as the ether lipid concentration was increased, totally disappearing at concentrations higher than 5 mol%. Small-angle X-ray diffraction and 31P-NMR confirmed that ET-18-OCH3 induced that the appearance of the inverted hexagonal HII phase was shifted towards higher temperatures completely disappearing at concentrations higher than 5 mol%. These results were used to elaborate a partial phase diagram and they were discussed as a function of the molecular action of ET-18-OCH3.

Summary: A variety of glycolipids were found in a collection of strains of Mycobacterium fortuitum and two patterns were established by thin-layer chromatography. A compound present in all the strains studied was identified as 2,3,4-triacyltrehalose and its overall structure determined by infrared spectroscopy, nuclear magnetic resonance spectroscopy and gas-liquid chromatography–mass spectrometry. Fatty acyl groups present in this molecule were identified as tetradecanoyl, hexadecenoyl, hexadecanoyl, octadecenoyl, octadecanoyl and a variety of 2-methyl-branched unsaturated (α-methyl, α,β-unsaturated) acyl groups. The 2-methyl-branched compounds ranged from 17 to 21 carbon atoms, the most abundant being 2-methyloctadecen-2-oyl. Trehalose was the only sugar detected in the glycolipid. This substance immunoreacted with IgG and IgM present in sera from tuberculosis patients, as demonstrated by an enzyme-linked immunosorbent assay.

The effect of edelfosine (1- O-octadecyl-2- O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3) on model membranes containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/cholesterol) was studied by several physical techniques. The sample POPC/SM (1:1 molar ratio) showed a broad phase transition as seen by DSC, X-ray diffraction, and 2H NMR. The addition of edelfosine to this sample produced isotropic structures at temperatures above the phase transition, as seen by 2H NMR and by 31P NMR. When cholesterol was added to give a POPC/SM/cholesterol (at a molar ratio 1:1:1), no transition was observed by DSC nor X-ray diffraction, and 2H NMR indicated the presence of a liquid ordered phase. The addition of 10 mol % edelfosine increased the thickness of the membrane as seen by X-ray diffraction and led to bigger differences in the values of the molecular order of the membrane detected at high and low temperatures, as detected through the M 1 first spectral moment from 2H NMR. These differences were even greater when 20 mol % edelfosine was added, and a transition was now clearly visible by DSC. In addition, a gel phase was clearly indicated by X-ray diffraction at low temperatures. The same technique pointed to greater membrane thickness in this mixture and to the appearance of a second membrane structure, indicating the formation of two separated phases in the presence of edelfosine. All of these data strongly suggest that edelfosine associating with cholesterol alter the phase status present in a POPC/SM/cholesterol (1:1:1 molar ratio) mixture, which is reputed to be a model of a raft structure. However, cell experiments showed that edelfosine colocalizes in vivo with rafts and that it may reach concentrations higher than 20 mol % of total lipid, indicating that the concentrations used in the biophysical experiments were within what can be expected in a cell membrane. The conclusion is that molecular ways of action of edelfosine in cells may involve the modification of the structure of rafts.

The effect of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), and six other analog asymmetric phosholipids on the physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes was studied using differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (31P NMR) and X-ray diffraction. DSC data revealed that, at concentrations of 40 mol% and higher, a new type of mixtures with higher Tc and narrower transitions appeared with all the asymmetric lipids studied. At very high concentrations of these lipids (50-80 mol%), destabilization was observed in the systems probably because of the formation of micelles or small vesicles. In all cases, the asymmetric lipids at concentrations of 40 mol% induced the formation of interdigitated structures in the lamellar gel phase, as deduced from X-ray diffraction. The asymmetric phospholipids were also added to 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) model membranes and DSC data revealed that the lipids primarily affected transition from the lamellar gel (Lβ) to the lamellar liquid crystalline (Lα) phase in two aspects: the transition temperature was reduced, and the transition itself became broader and smaller. The lamellar liquid crystalline (Lα) to inverted hexagonal phase (HII) transition was also affected, as detected by DSC and 31P NMR data. Increasing concentrations of the asymmetric lipids reduced the formation of inverted hexagonal phases, which were completely inhibited in the case of ET-18-OCH3. Since these compounds have been shown to have important biological actions through the plasma membrane, these results may help to understand the mechanism of action of these compounds. In addition these asymmetric lipids were tested for their capacity to induce cell apoptosis, and only ET-18-OCH3 was found to have a clear effect, thus suggesting that the apoptotic effect is not exerted through changes in the biophysical properties of model membranes. © 2006 Elsevier B.V. All rights reserved., This paper was supported by Grant 1FD97-0622 from the European Commission and Comisión Interministerial de Ciencia y Tecnología, Grant BFU2005-02482/BMC (to J.C.G.F.) from Dirección General de Investigación (Spain), Grant 00591/PI/04 (to J.C.G.F.) from Fundación Séneca (Comunidad Autónoma de Murcia, Spain), and Grant FIS04-0843 from Fondo de Investigación Sanitaria (to F.M.). C.G. and S.C.-G were supported by the Ramón y Cajal Program from the Ministerio de Educación y Ciencia of Spain.

Infrared spectroscopy was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic protein Bak (Bak-C) and the anti-apoptotic protein Bcl-2 (Bcl-2-C) when incorporated into different lipid vesicles. Whereas beta-pleated sheet was the predominant type of secondary structure of Bak-C in the absence of membranes, the same peptide adopted different structures depending on lipid composition when incorporated into membranes, with the predominance of the alpha-helical structure in the case of DMPC and other phospholipids, such as POPC and POPG. However, beta-pleated sheet was the predominant structure in other membranes containing phospholipids with longer fatty acyl chains and cholesterol, as well as in a mixture which imitates the composition of the outer mitochondrial membrane (OMM). Similarly, Bcl-2-C adopted a structure with a predominance of intermolecularly bound pleated beta-sheet in the absence of membranes, with alpha-helix as the main component in the presence of DMPC and POPG, but intermolecular beta-sheet in the presence of EYPC and cholesterol. Using ATR-IR, it was found that the orientation of the alpha-helical components of both domains was nearly perpendicular to the plane of the membrane in the presence of DMPC membranes, but not in EYPC or OMM membranes. (2)H NMR spectroscopy of DMPC-d(54) confirmed the transmembrane disposition of the domains, revealing that they broadened the phase transition temperature, although the order parameter of the C-D bonds was not affected, as might have been expected for intrinsic peptides. When all these results are taken together, it was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes, as happens in the membrane imitating the composition of the OMM, where the peptides were partially located outside the membranes.

Iturin A is a lipopeptide extracted from the culture media of Bacillus subtilis which shows a strong antifungal action. The interaction of iturin A with multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) induced structures which did not sediment during centrifugation. Electron microscopy after negative staining showed that, at 30 mol%, iturin A/DMPC vesicles were visible but smaller than those formed by pure DMPC. Thermograms of DMPC/iturinA obtained after differential scanning calorimetry, at low concentrations of iturin A, were interpreted as indicating the presence of two laterally separated phases, one formed by pure phospholipid and the other by lipopeptide-phospholipid complexes, these two separated phases being already detected even at low concentrations such as 2 mol%. Fluorescence quenching experiments showed that the D-Tyr residue of the lipopeptide was fully accessible to the aqueous medium, indicating that the polar part of iturin A is located outside of the membrane hydrophobic palisade. It was concluded that the membrane barrier properties are likely to be damaged in the area where the lipid complexes are accumulated, due to structural fluctuations, and this may be one of the bases of its biological activity. Iturin-A was also able to greatly destabilize dielaidoylphosphatidylethanolamine (DEPE) membranes in the fluid form, producing a new structure which had a poor correlation in X-ray diffraction, and in 31P NMR spectroscopy gave rise to a spectrum containing a double isotropic signal. Iturin A was shown to induce DEPE to adopt phases other than H(II) inverted hexagonal, underlining that this lipopeptide is capable of modifying the curvature of the membrane, which may also be important in explaining the tendency of iturin A to create small vesicles and which may be another of the bases of its biological activity.

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.

Curcumin is a polyphenol present in turmeric, a spice widely used in Asian traditional medicine and cooking. It has many and diverse biological effects and is incorporated in cell membranes. This paper describes the mode in which curcumin modulates the physical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dielaidyl-sn-glycero-3-phosphoetnanolamine (DEPE) multilamellar membranes. Curcumin disordered DPPC membranes at temperatures below T(c) as seen by DSC, FT-IR, (2)H NMR, WAXD, and SAXD. The decrease induced by curcumin in T(c) suggested that it is oriented in the bilayer with its main axis parallel to the acyl chains. Above T(c), too, curcumin introduced disorder as seen by infrared spectroscopy which showed that curcumin also alters the conformation of the polar group of DPPC, increasing the percentage of unhydrated C=O groups, but does not form hydrogen bonds with either the C=O group or the phosphate group of DPPC. Small angle X-ray diffraction showed a notable increase in the repeating spacings as a result of the presence of curcumin, suggesting the formation of a rippled phase. Increasing concentrations of curcumin progressively modified the onset and completion of the phase transition and also DeltaH up to a 6:1 DPPC/curcumin molar ratio. A further increase of curcumin concentration did not produce effects on the transition parameters, suggesting that there is a limit for the solubility of curcumin in DPPC. Additionally, when DEPE was used to test the effect of curcumin on the phospholipid polymorphism, it was found that the temperature at which the H(II) phase is formed decreased, indicating that curcumin favors negative curvature of the membrane, which may be important for explaining its effect on membrane dynamics and on membrane proteins or on proteins which may be activated through membrane insertion.