Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Influence of acylcarnitines of different chain length on pure and mixed phospholipid vesicles and on sarcoplasmic reticulum vesicles

Palmitoyl-, myristoyl- and lauroylcarnitine destabilize small unilamellar vesicles of 1,2-dipalmitoyl-n-glycero-3-phosphorylcholine (DPPC) and 1,2-dimyristoyl-n-glycero-3-phosphorylcholine (DMPC) into multilamellar liposomes. Their effect on the bilayer is dependent on the acyl chain length of the acylcarnitine, the ratio of the lengths of the acyl chains of the phospholipid and the acylcarnitine and the molar ratio of the phospholipid to acylcarnitine but not the absolute concentration of the acylcarnitine in the solute. Sarcoplasmic reticulum vesicles are broken down by each of the acylcarnitines at concentrations below their critical micellar concentrations (CMC). These three acylcarnitines stimulate the Mg2+, Ca2+-ATPase activity in SR-vesicles to a certain maximum, after which a net inhibition is observed. The maximum degree of stimulation depends highly on acyl chain length: the shorter the chain length, the more effective. In the same concentration range where the Mg2+, Ca2+-ATPase activity is increased, the net Ca2+-uptake is markedly decreased.     

Effect of acyl chain mismatch on the contact mechanics of two-component phospholipid vesicle during main phase transition

It has been recently demonstrated that acyl chain mismatch of phospholipid bilayer composed of a binary lipid mixture induces component formation on the lateral plane of the bilayer [Biophys. J. 83 (2002) 1820-1883]. In this report, the contact mechanics of unilamellar vesicles composed of binary dimyristoyl-phosphatidylcholine (DMPC)/dipalmitoyl-phosphocholine (DPPC) mixtures on fused silica and amino-modified substrates is simultaneously probed by confocal-reflectance interference contrast microscopy (C-RICM) and cross-polarized light microscopy during gel to liquid crystalline transition of the lipid bilayer. C-RICM results indicate that the average degree of vesicle deformation for DMPC-rich and DPPC-rich vesicles adhering on fused silica substrate is increased by 30% and 14%, respectively, in comparison with that in pure DMPC and DPPC vesicles. Also, lateral heterogeneity induced by acyl chain mismatch increases the average magnitude of adhesion energy in DMPC-rich and DPPC-rich vesicles of all sizes by 6.4 times and 2.3 times, respectively. Similar modulation of adhesion mechanics induced by carbon chain difference is obtained on amino-modified substrate. Most importantly, the thermotropic transition of the mixed bilayer from gel (below T(m)) to fluid phase (above T(m)) further exemplifies the effect of acyl chain mismatch on the increases of degree of vesicle deformation and adhesion energy.     

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.     

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Interaction of a synthetic peptide corresponding to the N-terminus of canine distemper virus fusion protein with phospholipid vesicles: a biophysical study

The F protein of canine distemper virus (CDV) is a classic type I glycoprotein formed by two polypeptides, F1 and F2. The N-terminal regions of the F1 polypeptides of CDV, measles virus and other paramyxoviruses present moderate to high homology, supporting the existence of a high conservation within these structures, which emphasises its role in viral-host cell membrane fusion. This N-terminal polypeptide is usually termed the fusion peptide. The fusion peptides of most viral fusion-mediating glycoproteins contain a high proportion of hydrophobic amino acids, which facilitates its insertion into target membranes during fusion. In this work we report on the interaction of a 31-residue synthetic peptide (FP31) corresponding to the N terminus of CDV F1 protein with phospholipid membranes composed of various phospholipids, as studied by means of various biophysical techniques. FTIR investigation of FP31 secondary structure in aqueous medium and in membranes resulted in a major proportion of alpha-helical structure which increased upon membrane insertion. Differential scanning calorimetry (DSC) showed that the presence of concentrations of FP31 as low as 0.1 mol%, in mixtures with L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dipalmitoylphosphatidylcholine (DPPC) and L-alpha-distearoylphosphatidylcholine (DSPC), already affected the thermotropic properties of the gel to liquid-crystalline phase transition. In mixtures with the three lipids, increasing the concentration of peptide made the pretransition to disappear, and lowered and broadened the main transition. This effect was slightly stronger as the acyl chain length of the phospholipid grew larger. In the corresponding partial phase diagrams, no immiscibilities or critical points were observed. FTIR showed that incorporation of 1 mol% of peptide in DPPC shifted the antisymmetric and symmetric CH2 stretching bands to higher values, indicating the existence of an additional disordering of the acyl chain region of the fluid bilayer. FTIR studies of the Cz=O stretching band indicated that incorporation of FP31 into phosphatidylcholine membranes produced a strong dehydration of the polar part of the bilayer. In mixtures with L-alpha-dielaidoylphosphatidylethanolamine (DEPE), increasing FP31 concentrations broadened and shifted to lower temperatures the lamellar to hexagonal-HII phase transition, indicating that this peptide destabilized the bilayer and promoted formation of the hexagonal-HII phase. The results are discussed in terms of lipid-peptide hydrophobic mismatch and its influence on the role of the N-terminal polypeptide of CDV F1 protein in viral membrane fusion.     

Interactions of angiotensin II non-peptide AT(1) antagonist losartan with phospholipid membranes studied by combined use of differential scanning calorimetry and electron spin resonance spectroscopy.

We used differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy to investigate the interactions of Losartan, a potent, orally active Angiotensin II AT(1) receptor antagonist with phospholipid membranes. DSC results showed that Losartan sensitively affected the chain-melting behavior of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. ESR spectroscopy showed that phosphatidylcholines spin-labeled at the 5-position of the sn-2 acyl chain (n-PCSL with n=5), incorporated either in DMPC or DPPC bilayers containing Losartan, were restricted in motion both in the gel and in the liquid-crystalline membrane phases, indicating a location of the antagonist close to the interfacial region of the phosphatidylcholine bilayer. At high drug concentrations (mole fraction >/= x=0.60), the decrease in chain mobility registered by 5-PCSL in fluid-phase membranes is smaller than that found at lower concentrations, whereas that registered by 14-PCSL is further increased. This indicates a different mode of interaction with Losartan at high concentrations, possibly arising from a location deeper within the bilayer. Additionally, Losartan reduced the spin-spin broadening of 12-PCSL spin labels in the gel-phase of DMPC and DPPC bilayers. As a conclusion, our study has shown that Losartan interacts with phospholipid membranes by affecting both their thermotropic behavior and molecular mobility.     

Cadmium-induced changes in the membrane of human erythrocytes and molecular models

The structural effects of cadmium on cell membranes were studied through the interaction of Cd(2+) ions with human erythrocytes and their isolated unsealed membranes (IUM). Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Cd(2+) induced shape changes in erythrocytes, which took the form of echinocytes. According to the bilayer couple hypothesis, this result meant that Cd(2+) ions located in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar headgroup and the acyl chain packing arrangements of the membrane phospholipid bilayer. Cd(2+) ions also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers the erythrocyte membrane, respectively. X-ray diffraction indicated that Cd(2+) ions induced structural perturbation of the polar headgroup and of the hydrophobic acyl regions of DMPC, while the effects of cadmium on DMPE bilayers were much milder. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles (LUV). All these findings point to the important role of phospholipid bilayers in the interaction of cadmium on cell membranes.     

Nuclear magnetic resonance and thermal studies on the interaction between salicylic acid and model membranes

DSC and (1H and 31P) NMR measurements are used to investigate the perturbation caused by the keratolytic drug, salicylic acid (SA) on the physicochemical properties of the model membranes. Model membranes (in unilamellar vesicular (ULV) form) in the present studies are prepared with the phospholipids, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA) and mixed lipid DPPC-DPPE (with weight ratio, 2.5:2.2). These lipids have the same acyl (dipalmitoyl) chains but differed in the headgroup. The molar ratio of the drug to lipid (lipid mixture), is in the range 0 to 0.4. The DSC and NMR results suggest that the lipid head groups have a pivotal role in controlling (i) the behavior of the membranes and (ii) their interactions with SA. In the presence of SA, the main phase transition temperature of (a) DPPE membrane decreases, (b) DPPA membrane increases and (c) DPPC and DPPC-DPPE membranes are not significantly changed. The drug increases the transition enthalpy (i.e., acyl chain order) in DPPC, DPPA and DPPC-DPPE membranes. However, the presence of the drug in DPPC membrane formed using water (instead of buffer), shows a decrease in the transition temperature and enthalpy. In all the systems studied, the drug molecules seem to be located in the interfacial region neighboring the glycerol backbone or polar headgroup. However, in DPPC-water system, the drug seems to penetrate the acyl chain region also.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Temperature-dependence of the solubilization of dipalmitoylphosphatidylcholine (DPPC) by the non-ionic surfactant Triton X-100, kinetic and structural aspects

Most of the studies on the solubilization of model membranes conducted thus far involved model membranes made of liquid-crystalline phospholipids. Relatively little is known on the influence of temperature and of the phase of the lipid bilayers on their solubilization by detergents. The aim of the present study was to gain knowledge about the temperature and phase dependence of the solubilization of phospholipid bilayers by the non-ionic detergent Triton X-100 (TR). Detailed investigation of the kinetics of the solubilization of dipalmitoylphosphatidylcholine (DPPC), as well as of palmitoyloleoylphosphatidylcholine (POPC) by TR at different temperatures reveals that: (i) solubilization of DPPC is a relatively slow process, especially below Tm. This means that in order to prevent misleading conclusions it is important to monitor the solubilization after a steady state is established. (ii) Both the steady state structure and size of DPPC/TR aggregates and the kinetics of solubilization depend on temperature. (iii) The TR concentration required for solubilization of POPC bilayers is an increasing function of temperature, although no phase change of bilayers occurs in the studied temperature range. (iv) Detailed studies of the temperature-induced changes of the aggregates present in DPPC/TR or POPC/TR mixtures suggest that the state of aggregation at any temperature above 23 degrees C represents equilibrium. By contrast, for DPPC/TR mixtures at 4 degrees C all the processes are very slow, which complicates the interpretation of results obtained through the common practice of studying "rafts" by investigating detergent-resistant membranes.     

Oriented 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/ganglioside membranes: a Fourier transform infrared attenuated total reflection spectroscopic study. Band assignments; orientational, hydrational, and phase behavior; and effects of Ca2+ binding.

Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy was used to elucidate the hydration behavior and molecular order of phospholipid/ganglioside bilayers. We examined dry and hydrated films of the gangliosides GM1, deacetyl-GM1, lyso-GM1, deacetyllyso-GM1, and GM3 and oriented mixed films of these gangliosides with 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) using polarized light. Analysis of the amide I frequencies reveals that the amide groups are involved in intermolecular interactions via hydrogen bonds of varying strengths. The tilt angle of the acyl chains of the lipids in mixed films was determined as a function of ganglioside structure. Deacetylation of the sialic acid in the headgroup has a stronger influence on the tilt angle than the removal of the ganglioside fatty acid. The phase behavior was examined by FTIR ATR spectroscopy and by differential scanning calorimetry (DSC) measurements on lipid suspensions. At the same molar concentration, lyso-gangliosides have less effect on changes of transition temperature compared to the double-chain analogs. Distinct differences in the amide band shapes were observed between mixtures with lyso-gangliosides and normal double-chain gangliosides. Determined from the dicroic ratio RATR, the orientation of the COO- group in all DMPC/ganglioside mixtures was found to be relatively fixed with respect to the membrane normal. In 4:1 mixtures of DMPC with GM1 and deacetyl-GM1, the binding of Ca2+ leads to a slight decrease in chain tilt in the gel phase, probably caused by a dehydration of the membrane-water interface. In mixtures of DMPC with GM3 and deacetyl-lyso-GM1, a slight increase in chain tilt is observed. The chain tilt in DMPC/lyso-GM1 mixtures is unchanged. Analysis of the COO- band reveals that Ca2+ does not bind to the carboxylate group of the sialic acid of GM1 and deacetyl-GM1, the mixtures in which a decrease in chain tilt was observed. Binding to the sialic acid was only observed for mixtures of DMPC with GM3, lyso-GM1, and deacetyl-lyso-GM1. Ca2+ obviously accumulates at the bilayer-water interface and leads to partial dehydration of the headgroup region in the gel as well as in the liquid-crystalline phase. This can be concluded from the changes in the amide I band shapes. With the exception of DMPC/deacetyl-GM1, the effects on the ester C==O bands are small. The addition of Ca2+ has minor effects on the phase behavior, with the exception of the DMPC/GM1 mixture.     

Interaction of a bacterial monorhamnolipid secreted by Pseudomonas aeruginosa MA01 with phosphatidylcholine model membranes

This work presents a biophysical study on the interactions of a monorhamnolipid (monoRL) produced by Pseudomonas aeruginosa MA01 with model phosphatidylcholine membranes. The molecular characterization of the biological activities, including the modulation of phospholipid membranes structure, of this monoRL biosurfactant is of importance for the validation of this particular Pseudomonas aeruginosa strain as a useful biosurfactant producer. The marked amphiphilic structure of monoRL is expected to result in strong interactions with the phospholipid constituents of membrane bilayers. Incorporation of monoRL into DMPC completely abolished the pretransition, and the main gel to liquid-crystalline phase transition was progressively broadened and shifted to lower temperatures, as observed by differential scanning calorimetry. Partial phase diagrams for DPPC and DSPC indicated near-ideal behavior. However, the DMPC diagram indicated fluid phase immiscibility. X-ray diffraction showed and apparent increase in d-value for DPPC containing monoRL, which might be the result of an effective increase in the bilayer thickness, or in the thickness of the hydration layer between bilayers. FTIR indicated that interaction of monoRL with the phospholipid acyl chains did not result in a large additional disordering of the acyl chain region of the fluid bilayer. Analysis of the CO stretching band of DPPC indicated an important effect of monoRL on the interfacial region of phosphatidylcholine bilayers, which might contribute to explain some of the biological activities of this glycolipid.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, ∼44% of phospholipids are left insoluble at 4 °C in bovine hippocampal membranes. Cholesterol depletion using methyl-β-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.     

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, approximately 44% of phospholipids are left insoluble at 4 degrees C in bovine hippocampal membranes. Cholesterol depletion using methyl-beta-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.     

Anomalous mixing of zwitterionic and anionic phospholipids with double-chain cationic amphiphiles in lipid bilayers.

High-sensitivity scanning calorimetry has been used to examine the thermotropic behavior of mixtures combining dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine (DPPE) and O-methylphosphatidic acid (DPPA-OMe) with the double-chain cationic amphiphiles N,N-dihexadecyl-N,N- dimethylammonium chloride (DHDAC), 1,2-dipalmitoyloxy-3-(trimethylammonio)propane (DPTAP) and the corresponding monomethylated tertiary amino compounds (DHMMA-H+ and DPDAP-H+). At physiological ionic strength, mixtures of these cationic amphiphiles with the anionic phospholipid DPPA-OMe can show gel-to-liquid-crystalline phase transitions at considerably higher temperatures than do either of the pure components. Surprisingly, binary mixtures of DPPC and these cationic amphiphiles also show strongly nonideal mixing, with phase diagrams exhibiting pronounced maxima in their solidus and liquidus curves. Similar behavior is not observed for mixtures of DPPC with DPPA-OMe, which closely resembles DPTAP and DPDAP-H+ in backbone configuration and polar headgroup size. The present results suggest that perturbation of the orientation of the phosphatidylcholine headgroup by cationic amphiphiles, as demonstrated previously by Seelig and co-workers (Biochemistry 28 [1989], 7720-7728), can significantly affect the thermotropic behavior of phospholipids such as DPPC. Such effects may exert a generally important (though not always easily recognizable) influence on the organization and thermotropic behavior of systems where zwitterionic phospholipids are combined with charged bilayer-associated molecules.     

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure.     

Membrane fluidity as affected by the organochlorine insecticide DDT

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of DDT with model and native membranes. DDT decreases the phase transition midpoint temperature (Tm) of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), and broadens the thermotropic profile of the transition. The effects of DDT are concentration dependent and are more pronounced in bilayers of short-chain lipids, e.g., DMPC. The insecticide fails to alter DPH polarization in the fluid phase of the above lipids. Similar effects were observed in binary mixtures of DMPC plus DPPC. Furthermore, DDT alters the single broad transition of the equimolar mixture of DMPC plus DSPC into a biphasic transition. The lower temperature component has a midpoint at 25 degrees C, i.e., a value close to the Tm of DMPC. DDT inhibits to some extent the cholesterol-induced ordering in DMPC bilayers and high cholesterol concentrations (greater than or equal to 30 mol%) do not prevent insecticide interaction, conversely to the effect observed for lindane (Antunes-Madeira, M.C. and Madeira, V.M.C. (1989) Biochim. Biophys. Acta 982, 161-166). Apparently, the bilayer order is not disturbed by DDT in fluid native membranes of mitochondria and sarcoplasmic reticulum, but moderate disordering effects are noticed in membranes enriched in cholesterol, namely, brain microsomes and erythrocytes.