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The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca2+-independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCe, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature.

A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and two cationic peptides derived from its sequence, ΔM1 (+5) and ΔM2 (+9) is reported in this work. The influence of charge on the interactions between peptides and membranes and its effect on phase were studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that ΔM2 peptide showed the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol (PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to determine lipid segregation in the presence of peptides. When WT and ΔM1 bound to model membrane containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, ΔM2 peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also suggested that the presence of ΔM1 and ΔM2 peptides increased lipid carbonyl group hydration in DMPG membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in the presence of WT and ΔM1 peptides in DMPC/DMPG. Overall the increase in positively charged residues favors the interaction of the peptides with the negatively charged membrane and its perturbation.

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.

Curcumin modulates the activity of protein kinase Cα (PKCα) when assayed in the presence of vesicles including phosphatidylcholine, phosphatidylserine and diacylglycerol. Increasing concentrations of curcumin progressively increased PKCα activity at concentrations lower than 20 μM, but at higher concentrations of curcumin the activity decreased although, at concentrations of curcumin of up to 100 μM the activity was always higher than the basal one (in the absence of curcumin). The maximum activity was reached at 3 μM curcumin, at 20 and 30 mol% of phosphatidylserine, 10 μM Ca2+ and 2 mol% diacylglycerol. The same type of modulation was observed when changing the concentration of phosphatidylserine, diacylglycerol and Ca2+. No effect of curcumin was found when the activity was assayed in the presence of Triton X-100 mixed micelles which included phosphatidylserine and diacylglycerol, indicating that the effect of curcumin was membrane-dependent. The pattern of binding of PKCα to membrane vesicles as a function of curcumin concentration closely correlated with the pattern of activating effect. It was concluded that the effect of curcumin on PKCα activity was related to its effect on the membrane, which may modulate the binding of the enzyme to the membrane.

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 effect of alpha-tocopherol on the Ca(2+)-induced fusion of large unilamellar phosphatidylserine vesicles has been investigated. Mixing of aqueous vesicle contents was followed continually with the terbium-dipicolinic acid (Tb-DPA) assay, while the dissociation of preencapsulated Tb-DPA complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure phosphatidylserine and phosphatidylserine containing 2, 5, and 10 mol% of alpha-tocopherol were employed at different Ca2+ concentrations. The presence of low amounts of alpha-tocopherol decreased the initial rate of fusion without changing the Ca2+ threshold concentration. The reduction of the initial rate of fusion was proportional to the amount of alpha-tocopherol present in the bilayer. An alpha-tocopherol concentration-dependent decrease of both the initial rate and the final extent of release of vesicle contents was also observed. This effect was more evident as more alpha-tocopherol was incorporated in the bilayer, so that in the presence of 10 mol% of alpha-tocopherol no significant release was observed after 5 min. The stabilization of the vesicular structure exerted by alpha-tocopherol was responsible for the apparent increase of the fluorescence intensity of the Tb-DPA complex at later stages of the process. The results reflect a perturbation of the membrane by low concentrations of alpha-tocopherol which may account for a number of biological effects of this vitamin, not related to its antioxidant role.

Ritodrine inhibits the steady-state Ca 2+ -ATPase activity of isolated sarcoplasmic reticulum vesicles in a dose-dependent manner, The observed K 0.5 value for inhibition (∼3 mM) gives proof of a low-affinity interaction. Ritodrine also interferes with the steady-state Ca 2+ transport ability decreasing the maximal rate without modification of the Ca 2+ or ATP affinity for the enzyme, This is consistent with an absence of competition for the transport and the catalytic sites. Analysis of the catalytic and transport cycle shows that: (i) ritodrine inhibits the phosphorylation partial reaction, This is supported by pre-steady-state kinetic experiments of Ca 2+ transport and also by the temperature dependence of the phosphoenzyme level. (ii) At high ritodrine concentrations the dephosphorylation step becomes rate-limiting. This is suggested by the biphasic profile (V-shape) of phosphoenzyme accumulation at different ritodrine concentrations, This was also confirmed by chase experiments of radioactive phosphoenzyme decomposition at steady state. These data reveal a complex pattern of inhibition involving two sites for interaction with low and high ritodrine concentrations. It is envisaged that ritodrine does not exert any direct effect on the smooth muscle sarcoplasmic reticulum Ca 2+ -ATPase when used in the treatment of preterm birth.

The role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes was investigated in vitro, monitoring the mixing of membrane lipids with the octadecyl rhodamine B fluorescence dequenching assay. Polylysine was able to induce fusion of chromaffin granule ghosts with plasma membrane vesicles in the absence of Ca2+. This fusion was maximal at 1.6 μM polylysine and the kinetics were dependent on the amount of plasma membrane added. Polylysine lowered the Ca2+ threshold concentration for inducing fusion, increased the extent of fusion at a given Ca2+ concentration and acted synergistically with Ca2+ when added prior to the cation. Removal of membrane proteins by trypsinization of chromaffin granule ghost and/or plasma membranes increased the extent of polylysine induced fusion. Incorporation of diacylglycerol into the plasma membrane promoted Ca2+-induced fusion. Chromaffin granule ghosts were induced to fuse with model membranes of different complexities from one resembling the inner monolayer of the erythrocyte membrane to one composed solely of pure phosphatidylserine. The characteristics of the fusion with model membranes were qualitatively similar to that observed with target plasma membrane, although differences in kinetics and stoichiometries were found. Interestingly, considerably low (μM) Ca2+ concentrations were able to induce fusion of chromaffin granule ghosts with diacylglycerol containing phosphatidylserine vesicles in the presence of polylysine. Our results indicate that acidic phospholipid-like phosphatidylserine may have an important role in the process of fusion and suggest that diacylglycerol, as a destabilizing lipid, may have a role by itself in promoting exocytosis in chromaffin cells.

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.

The effect of the association of alpha-tocopherol (vitamin E) with single chain or very asymmetric phospholipids has been studied by 31P nuclear magnetic resonance, Fourier transform infrared spectroscopy, and light microscopy. Our results demonstrate that alpha-tocopherol stabilizes and forms bilayer structures in systems composed of either 1-palmitoyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, or 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/1-palmitoyl-sn-glycero-3-phosphocholine, whereas in the absence of alpha-tocopherol all these systems produce micellar structures. The free hydroxyl group of alpha-tocopherol is shown to be important in stabilizing the bilayer structure by comparing the effects produced by an analog of alpha-tocopherol, alpha-tocopheryl acetate, which has the hydroxyl group blocked by an acetyl group. It is suggested that the complementary shapes of alpha-tocopherol and the asymmetric phospholipids may be the reason for the stabilization of the bilayer structure.

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 m m phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 m m surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 m m phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a “shoulder” appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.

Coenzymes Q10 and Q3 are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ10 and CoQ3 reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.

Purified F1-ATPase is slowly inactivated by interaction, in a preincubation medium, with its substrate MgATP. Interaction with Mg2+ before addition of ATP to the preincubation medium is essential to induce the inactivation. This inactivation is different from other Mg2+-induced inhibitions previously described. Free ATP concentration is implicated in the inactivation process and a linear relationship can be established between this concentration and the number of turnovers which are necessary for total inactivation. ITP, 2′-dATP, and GTP can also induce inactivation. Although ITP and GTP are hydrolyzed at a lower rate than ATP and 2′-dATP, they induce inactivation after a smaller number of turnovers than the latter. This process closely follows a kinetics of the type described for suicide enzymes. A reaction scheme is suggested and discussed.