Role of peptide structure in lipid-peptide interactions: nuclear magnetic resonance study of the interaction of pentagastrin and [Arg4]pentagastrin with dimyristoylphosphatidylcholine

Complexes formed between dimyristoylphosphatidylcholine (DMPC) and the peptide pentagastrin or [Arg4]pentagastrin were examined by 31P- and 2H-NMR. The cationic [Arg4]pentagastrin produces larger changes in the lipid NMR spectra than does the anionic pentagastrin. 31P-NMR spectra of DMPC with [Arg4]pentagastrin below the phase transition exhibits two components one of which is motionally restricted compared with the pure lipid. The exchange between these two lipid domains is slow on the millisecond time scale. The interactions between this peptide and phospholipid are diminished above the melting temperature of the complex. The 2H-NMR spectra of DMPC which had been labelled in a choline methylene group is also affected more by the [Arg4]pentagastrin than by pentagastrin. In the presence of [Arg4]pentagastrin, even above the lipid phase transition, an additional doublet with a smaller quadrupole splitting is observed. These results clearly demonstrate the importance of peptide charge in determining the effects of peptides on lipid bilayers.     

Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides

The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.     

The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.

The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.     

Interaction of (+)-catechin with a lipid bilayer studied by the spin probe method

The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.     

A comparison of the interaction of glucagon, human parathyroid hormone-(1-34)-peptide and calcitonin with dimyristoylphosphatidylglycerol and with dimyristoylphosphatidylcholine

The interaction of glucagon, human parathyroid hormone-(1-34)-peptide and salmon calcitonin with dimyristoylphosphatidylglycerol (DMPG) and with dimyristoylphosphatidylcholine (DMPC) was studied as a function of pH and temperature. The effect of lipid on the secondary structure of the peptide was assessed by circular dichroism and the effect of the peptide on the phase transition properties of the lipid was studied using differential scanning calorimetry. Some peptides interact more strongly with anionic than with zwitterionic phospholipids. This does not require an overall positive charge on the peptide. Increased thermal stability is observed in complexes formed between cationic peptides and anionic lipids. Particularly marked effects of glucagon and human parathyroid hormone-(1-34)-peptide on the phase transition properties of DMPG at pH 5 have been observed. The transition temperature is raised over 10 degrees C at a lipid/peptide molar ratio of less than 30:1 and the transition enthalpy is increased over 2-fold. These effects do not occur with any basic peptide and were not observed with metorphinamide, molluscan cardioexcitatory neuropeptide or myelin basic protein. The results demonstrate that certain peptides can affect the phase transition properties of lipids in a manner similar to divalent cations. The overall hydrophobicities of these peptides can be evaluated by their partitioning between aqueous and organic solvents. None of the above three peptide hormones partition into the organic phase. However, a closely related peptide, human calcitonin, does exhibit substantial partitioning into the organic phase. Nevertheless, human calcitonin has a weaker interaction with both DMPC and DMPG than does salmon calcitonin. The effects of human calcitonin on the phase transition of DMPC are qualitatively different from those of salmon calcitonin in that the human form more readily eliminates the pretransition but causes less change in the main transition. Like overall charge, overall hydrophobicity is not an overwhelming factor in determining the ability of peptides to interact with phospholipids but rather more specific interactions are required for strong complexes to form.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Concentration of oxygen in lipid bilayers using a spin-label method.

The concentration of oxygen in the hydrocarbon region of lipid bilayer has been determined using a novel electron spin resonance (ESR) nitroxide-radical spin-probe method. For dimyristoylphosphatidylcholine (DMPC), the partition coefficient above the main transition temperature is approximately 3. Rapid decrease to 0.2 occurs below the pretransition temperature indicating exclusion of oxygen in the crystalline phase. The differences of molar free energy, enthalpy, and entropy of mixing between water and lipid have been determined for each phase.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of peptide structure in lipid-peptide interactions: a fluorescence study of the binding of pentagastrin-related pentapeptides to phospholipid vesicles

The binding of pentagastrin and three other structurally related pentapeptides to phospholipid vesicles has been studied by fluorescence spectroscopy. The fluorescence of the tryptophan residues of these peptides exhibits an increased quantum yield upon binding to phospholipid vesicles. This is accompanied by a blue shift of the maximum emission, indicative of the incorporation of the tryptophan residue into a more hydrophobic environment. The affinity of the peptides for a zwitterionic phospholipid, dimyristoylphosphatidylcholine (DMPC), increases in the following order: N-t-Boc-beta-Ala-Trp-Met-Gly-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2. Comparison of the interaction of these various peptides with this phospholipid indicates that although the interaction is largely of hydrophobic nature, the structure of the polar amino acids and their electrostatic charge have significant influence on the nature of the bindings. In addition, the sequence of polar and apolar amino acids appears to be of importance. The higher affinity for DMPC of N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 as compared to its "reversed" analogue N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2 suggests that the ability of the peptides to fold into amphiphatic structures can enhance their lipid binding affinity. For all peptides the interaction with DMPC is greater at 8 degrees C, i.e., below the lipid phase transition temperature, than at 40 degrees C, i.e., above the lipid phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Precision parameters from spin-probe studies of membranes using a partitioning technique. Application to two model membrane vesicles

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.     

Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution

Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed (31)P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE (T(H)) was observed for penetratin and an increase for RL16. Contrary to penetratin, (31)P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.     

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.     

Effect of staphylococcal delta-lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier transform infrared spectroscopic, and X-ray diffraction studies

We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our 31P NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are studied, the isotropic component of the 31P NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase 31P NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic 31P NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D(2)O. However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

ESR studies on the effect of cholesterol on chlorpromazine interaction with saturated and unsaturated liposome membranes

In this study, the effects of chlorpromazine (CPZ) on lipid order and motion in saturated (DMPC, DMPG) and unsaturated (SOPC) liposome membranes were investigated by electron spin resonance (ESR) spin labeling technique. We have shown that above the main phase transition temperature of membrane lipids (T(M)), CPZ slightly increases lipid order in membranes without cholesterol, whereas below T(M) it has a strong opposite effect. Addition of 30 mol% of cholesterol into DMPC and SOPC membranes changes significantly the CPZ effects both above and below T(M). Additionally, above T(M), the ordering effect of CPZ on pure SOPC membrane is stronger at pH 7.4 than at pH 9.0, whereas below T(M), as well as in the presence of cholesterol, pH does not seem to play a role in CPZ effect on both membranes. Because of the strong influence of membrane composition on CPZ effect on membranes, the use of cholesterol as a marker of CPZ photosensitized reactions has been discussed.     

Pentachlorophenol-induced change of zeta-potential and gel-to-fluid transition temperature in model lecithin membranes

We have determined zeta-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The zeta-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in zeta-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pretransition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pretransition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in delta Tt is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the delta Tt from photometric studies with the density of adsorbed PCP derived from zeta-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Thermodynamics of partitioning and efflux of phenothiazines from liposomes

Summary The partitioning of nine phenothiazines between dimyristoylphosphatidylcholine (DMPC) liposomes and 0.9% wt/vol saline at pH 6 has been studied both below and above the phase transition temperature (T _c ) of the phospholipid. Higher partitioning was observed aboveT _c . Both the entropy and enthalpy of partitioning were positive below and aboveT _c , and a linear relationship between the entropy and enthalpy has been derived. In general, the partitioning and transport of alkylaminophenothiazines in DMPC liposomes over the temperature range of 5 to 40°C is entropically controlled. The entropies and enthalpies of partitioning of various groups in the phenothiazine structure have been calculated.No relationship was found between particle size of the DMPC liposomes and the equilibrium partition coefficient at 25°C. However, the particle size of liposomes did increase with increasing acyl chain length of the phospholipid.Using differential scanning calorimetry, the enthalpy and entropy of transition of the DMPC liposomes in the absence and presence of phenothiazines has been calculated. The temperature dependence of the first-order rate constant of trimeprazine tartrate transport in DMPC liposomes was investigated and was found to be maximum at theT _c of the phospholipid.     

Effect of phase transitions in hydrated 1,2-dipalmitoylphosphatidylethanolamine bilayers on the spin probe order parameter

The ‘main’ phase transition L_β→L_α of hydrated 1,2-dipalmitoylphosphatidylethanolamine (DPPE) bilayers in excess water affects the ESR order parameter S₃₃ of N-cetyl-N,N-dimethyl-N-tempoylammonium bromide (CAT-16), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) spin probes. The ‘pretransition’ and ‘subtransition’ suggested to occur in hydrated DPPE by Chowdhry et al. [(1984) Biophys. J. 45, 901–904] and Silvius et al. [(1986) Biochemistry 25, 4249–4258], respectively, affect exclusively the S₃₃ of CAT-16, but not that of 5-DSA and 16-DSA spin probes. The subtransition occurs about 15 ± 1°C below the main transition.