Modulation of polymorphic properties of dielaidoylphosphatidylethanolamine by the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine.

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.

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.

Activation of protein kinase C alpha by lipid mixtures containing different proportions of diacylglycerols.

Lipid activation of protein kinase C alpha (PKC alpha) was studied using a model mixture containing POPC/POPS (molar ratio 4:1) and different proportions of either DPG or POG. The lipid mixtures containing DPG were physically characterized by using different physical techniques, and a phase diagram was constructed by keeping a constant POPC/POPS molar ratio of 4:1 and changing the concentration of 1,2-DPG. The phase diagram displayed three regions delimited by two compounds: compound 1 (CO(1)) with 35 mol % of 1,2-DPG and compound 2 (CO(2)) with 65 mol % of 1,2-DPG. PKC alpha activity was assayed at increasing concentrations of 1,2-DPG, maximum activity being reached at 30 mol % 1,2-DPG, which decreased at higher concentrations. Maximum activity occurred, then, at concentrations of 1,2-DPG which corresponded to the transition from region 1 to region 2 of the phase diagram. It was interesting that this protein was maximally bound to the membrane at all DPG concentrations. Similar results were observed when the enzyme was activated by POG, when a maximum was reached at about 10 mol %. This remained practically constant up to 50 mol %, about which it decreased, the binding level remaining maximal and constant at all POG concentrations. The fact that in the assay conditions used maximal binding was already reached even in the absence of diacylglycerol was attributed to the interaction of the C2 domain with the POPS present in the membrane through the Ca(2+) ions also present. To confirm this, the isolated C2 domain was used, and it was also found to be maximally bound at all DPG concentrations and even in its absence. Since the intriguing interaction patterns observed seemed to be due then to the C1 domain, the PKC alpha mutant D246/248N was used. This mutant has a decreased Ca(2+)-binding capacity through the C2 domain and was not activated nor bound to membranes by increasing concentrations of DPG. However, POG was able to activate the mutant, which showed a similar dependence on POG concentration with respect to activity and binding to membranes. These data underline the importance of unsaturation in one of the fatty acyl chains of the diacylglycerol.

The cancer chemopreventive agent resveratrol is incorporated into model membranes and inhibits protein kinase C alpha activity.

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.

A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers.

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.

Correlation between the effect of the anti-neoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase Calpha.

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 Calpha (PKCalpha) 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.

Influence of the physical state of the membrane on the enzymatic activity and energy of activation of protein kinase C alpha.

The activation of protein kinase C alpha was studied by using a lipid system consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) (molar ratio 4:1) and different proportions of 1-palmitoyl-2-oleoyl-sn-glycerol (POG). The phase behavior of the lipidic system was characterized by using differential scanning calorimetry and 31P NMR, and a phase diagram was elaborated. The results suggested the formation of two diacylglycerol/phospholipid complexes, one at 15 mol % of POG and the second at 30 mol % of POG. These two complexes would define the three regions of the phase diagram: in the first region (concentrations of POG lower than 15 mol %) there is gel-gel immiscibility at temperatures below that of the phase transition between C1 and pure phospholipid, and a fluid lamellar phase above of the phase transition. In the second region (between 15 and 30 mol % of POG), gel-gel immiscibility between C1 and C2 with fluid-fluid immiscibility was observed, while inverted hexagonal HII and isotropic phases were detected by 31P NMR. In the third region (concentrations of POG higher than 30 mol %), gel-gel immiscibility seemed to occur between C2 and pure POG along with fluid-fluid immiscibility, while an isotropic phase was detected by 31P NMR. When PKC alpha activity was measured, as a function of POG concentration, maximum activity was found at POG concentrations as low as 5-10 mol %; the activity slightly decreased as POG concentration was increased to 45 mol % at 32 degrees C (above Tc) whereas activity did not change with increasing concentrations of POG at 5 degrees C (below Tc). When the activity was studied as a function of temperature, at different POG concentrations, and depicted as Arrhenius plots, it was found that the activity increased with increasing temperatures, showing a discontinuity at a temperature very close to the phase transition of the system and a lower activation energy at the upper slope of the graph, indicating that the physical state of the membrane affected the interaction of PKC alpha with the membrane.