Influence of Ca2+ and Mg2+ on the thermotropic behaviour and permeability properties of liposomes prepared from dimyristoyl phosphatidylglycerol and mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine.

Calorimetric experiments showed a marked effect of Ca2+ and Mg2+ on the thermotropic behaviour of dimyristoyl phosphatidylglycerol. 2. Concentrations of Ca2+ and Mg2+ lower than 1 ion to 2 molecules of phosphatidylglycerol produced a shift of the phase transition to higher temperatures and an increase in the enthalpy change which is consistent with a closer packing of the lipid molecules in the liposomes. 3. Above the 1:2 ratio, freeze-fracture electron microscopy demonstrated typical "crystal" structures both in the presence of Ca2+ and Mg2+. In the presence of Mg2+ a metastable behaviour was noticed in the calorimetric experiments. 4. A Ca2+- and Mg2+-induced shift in the transition temperature and an increase in the enthalpy change was also observed in a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine. However, these mixed samples remained liposomal in structure at any concentration of the divalent ions. 5. Liposomes prepared from a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the absence of divalent cations are permeable in the range 10-50 degrees C. Bilayers of mixtures neutralized by Ca2+ or Mg2+ were demonstrated to be completely impermeable to K+, except in the vicinity of the phase transition. 6. The leak of ions from liposomes of a 1:1 mixture of dimyristoyl phosphatidylglycerol and dimyristoyl phosphatidylcholine in the vicinity of the phase transition temperature was considerably less in the presence of Ca2+ than in the presence of Mg2+. 7. It is concluded that there is a correlation between the calorimetric data and the permeability properties of dimyristoyl phosphatidylglycerol-containing bilayers with respect to the influence of Ca2+ and Mg2+.     

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin-spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.     

Smoothed acyl chain orientational order parameter profiles in dimyristoylphosphatidylcholine-distearoylphosphatidylcholine mixtures: a 2H-NMR study.

The accommodation of chain-length mismatch in liquid crystal phase bilayers was examined by using deuterium nuclear magnetic resonance to obtain smoothed orientational order parameter profiles for acyl chains of both components in binary lipid mixture bilayers. Mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) covering a range of compositions were prepared with either DSPC acyl chains or DMPC acyl chains perdeuterated. Orientational order parameters in the plateau regions of the smoothed profiles for both components were found to increase smoothly with increasing DSPC concentration. The orientational order parameters in the DSPC-smoothed profile were found to be slightly higher than corresponding values for DMPC over a wide range of bilayer composition. The shapes of the smoothed profiles for both components were found to be sensitive to bilayer composition. At low DSPC concentration, DSPC methylene deuterons near the bilayer center display a secondary plateau at low orientational order. At high DSPC concentration, the plateau of the DMPC-smoothed profile is stretched slightly. The concentration dependence of the smoothed profiles at low DSPC concentration appears to be consistent with a picture in which the last few segments of the DSPC chain cross the bilayer midplane, on average, but remain very disordered.     

Hydration force parameters of phosphatidylcholine lipid bilayers as determined from 2H-NMR studies of deuterated water.

The continuous decrease of the quadrupolar splitting of deuterated water interacting with phosphocholine lipid bilayers with growing water concentration is analyzed as a function of the water activity. From the apparent linear dependence on water activity a measure for hydration forces is obtained. The forces calculated are in the range of published data using sorption isotherms and osmotic stress technique in combination with SAXS. A simple interaction potential which includes orientational order of water adsorbed on surfaces gives a physical base for these findings. Therefore, deuterium NMR may become a powerful tool for hydration force analysis complementing well-known methods.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.     

Use of deuterated phospholipids in Raman spectroscopic studies of membrane structure. I. Multilayers of dimyristoyl phosphatidylcholine (and its -d54 derivative) with distearoyl phosphatidylcholine

The temperature dependence of the Raman spectrum has been studied for binary phospholipid mixtures of dimyristoyl phosphatidylcholine (and its chain deuterated -d54 derivative) with distearoyl phosphatidylcholine. Two distinct melting regions are observed for the 1 : 1 mole ratio mixture. The use of deuterated phospholipid permits the identification of the lower (approximately 22 degrees C) transition with primarily the melting of the shorter chain component, and the higher (approximately 47 degrees C) transition primarily with the melting of the longer chains. The C-H stretching vibrations of the distearoyl component respond to the melting of the dimyristoyl component, an apparent consequence of alterations in the lateral interactions of the distearoyl chains. These changes in the C-H spectral region suggest that phase separation does not occur in the gel state for this system. The results are in reasonable accord with recent calorimetric studies (Mabrey, S. and Sturtevant, J.M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3862-3866). The feasibility of using deuterated phospholipids to monitor the conformation of each component in a binary phospholipid mixture is demonstrated.     

Influence of staphylococcal delta-toxin on the phosphatidylcholine headgroup as observed using 2H-NMR.

The interaction of the 8-toxin peptide isolated from Staphylococcus aureus with the headgroup region of lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy. At relatively low peptide/lipid ratios (P/L < 0.10), all 2H- and 31P-NMR spectral lineshapes at 25 degrees C were indicative of a single population of liquid-crystalline lipids in a bilayer arrangement. At these P/L ratios, delta-toxin had only marginal effects on the size of the quadrupole splitting measured from POPC labelled at either the alpha-methylene (POPC-alpha-d2) or the beta-methylene segment (POPC-beta-d2) of the choline headgroup and, similarly small effects on the magnitude of the chemical shift anisotropy (CSA) of the 31P-NMR spectrum. With increasing amounts of delta-toxin (0.10 < P/L < 0.15) the size of the 2H quadrupole splitting from POPC-alpha-d2, as well as the magnitude of the 31P-CSA, decreased progressively and rapidly. The quadrupole splitting from POPC-beta-d2, however, remained relatively unaffected. At yet higher levels of delta-toxin (P/L > 0.15), all 2H- and 31P-NMR spectra indicated the presence of multiple lipid populations experiencing varying degrees of increased conformational disordering. The spectral lineshapes of these apparently nonbilayer spectral components reverted to bilayer-type lineshapes upon lowering the measuring temperature to 5 degrees C. At the utmost highest level of delta-toxin measured here (P/L = 0.20), all 2H- and 31P-NMR spectra consisted of a single, broad, apparently nonbilayer-type component, indicative of hindered but virtual isotropic motional averaging of the POPC headgroups. In this case no reversion to bilayer-type spectra could be obtained by decreasing the temperature. We could obtain no evidence that the conformation of the choline headgroup of POPC was responding to any specific influence of delta-toxin on bilayer surface electrostatics.     

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.     

Conformational changes of the phosphatidylcholine headgroup due to membrane dehydration. A 2H-NMR study

The influence of hydration on the orientation of the phosphocholine dipole in bilayer membranes was studied with nuclear magnetic resonance. The phosphocholine headgroup of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was deuterated at the two methylene segments. Phosphorus-31 and deuterium nuclear magnetic resonance measurements were made as a function of hydration in the range of 10-70 wt.% H2O revealing a distinct change in the alignment of the phosphocholine headgroup. With decreasing hydration the N+ end of the phosphocholine head group dipole moves closer to the hydrocarbon layer. The conformational change induced by the loss of water molecules at the membrane surface is qualitatively similar to that observed upon addition of polyhydroxyl compounds.     

Interaction of toposome from sea-urchin yolk granules with dimyristoyl phosphatidylserine model membranes: a 2H-NMR study

The yolk granule is the most abundant membrane-bound organelle present in sea urchin eggs and embryos. The major protein component of this organelle, toposome, accounts for approximately 50% of the total yolk protein and has been shown to be localized to the embryonic cell surface. Extensive characterization in several laboratories has defined a role for toposome in mediating membrane-membrane interactions. The current study expands the analysis of toposome-membrane interaction by defining toposome-induced changes to the lipid bilayer. The effect of toposome on the biophysical properties of phosphatidyl serine (PS) multibilayers was investigated using deuterium nuclear magnetic resonance and perdeuterated dimyristoyl PS (DMPS-d(54)). Toposome was found to have little effect on DMPS-d(54) chain orientational order in both the gel and liquid-crystalline phases. Timescales for DMPS-d(54) reorientation were investigated using quadropole echo decay. Echo decay times were sensitive to toposome in the liquid-crystalline phase but not in the gel phase. Additional information about the perturbation of bilayer motions by toposome was obtained by analyzing its effect on the decay of Carr-Purcell-Meiboom-Gill echo trains. Collectively, these results suggest that toposome interacts peripherally with DMPS bilayers and that it increases the amplitude of lipid reorientation, possibly through local enhancement of bilayer curvature.     

Detergent properties of apolipoprotein A1 in micellar complexes with dimyristoyl phosphatidylcholine

Bilayer micellar complexes of the human plasma apolipoprotein A1 with dimyristoyl phosphatidylcholine were prepared under kinetically controlled conditions. Detergent-like properties of Apo A1 in the complexes were expressed in terms of delta SA1 parameter (surface area of mixed micelle per an Apo A1 molecule). Analysis of our and earlier published data showed the correlation of the delta SA1 parameter with the stoichiometry of complexes. Changes of detergent-like properties were caused by cross-linking or proteolysis of Apo A1.     

Exchange and aggregation in dispersions of dimyristoyl phosphatidylcholine vesicles containing myristic acid

Processes occurring in dispersions of dimyristoyl phosphatidylcholine containing myristic acid have been studied by light scattering of dilute dispersions (concn. ≤ 1 mg/ml) at temperatures above and below the phase transition temperatures of these dispersions. The transition temperatures increase with increasing mol fraction of myristic acid. Above these temperatures, vesicles with different mol fractions of myristic acid exchange lipid molecules. The exchange process leads to vesicles having phase transition temperatures and radii, which are both intermediate between the initial transitions and radii, respectively. In contrast with the observations above the phase transitions, it was found that when dimyristoyl phosphatidylcholine/myristic acid vesicles were cooled to a few degrees below the phase transition, larger particles were formed. These observations are consistent with a mechanism consisting of vesicle aggregation followed by fusion of the aggregated vesicles. The aggregation process is of second order in the vesicle concentration, and its rate increases with increasing mol fraction of myristic acid.     

Human C-apolipoproteins promote hydrolysis of dimyristoyl phosphatidylcholine by snake venom phospholipase A2

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10^?5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [³H]thymidine incorporation. Proliferation is completely restored by 5 × 10^?5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10^?5 M each, fail to re-establish proliferation but produce an isotropic dilution of [³H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min^?1.10^?6 cells).     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.     

Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length

FT-IR spectroscopy has been used to evaluate the acyl chain conformational ordering of DMPC, DMPE, DMPA (pH 6 and 12), DMPG (pH 1 and 7), and DPPC, DPPE, DPPA (pH 6). The frequencies of the symmetric and antisymmetric methylene stretching vibrations were determined as a function of temperature. In the liquid-crystalline phase the frequencies show a qualitative dependence on the amount of chain disorder. Quantitative data for trans-gauche isomerization were obtained from the integral intensities of the conformation sensitive methylene wagging absorptions at ca. 1368 cm^–1 (gtg and gtg sequences), 1356 cm ^–1 (double gauche) and 1342 cm^–1 (end gauche). The integral band intensities were converted to the number of gauche conformers per acyl chain using the calibration factors published by Senak et al. (1991). At 69°C the highest number of gauche conformers excluding contributions from single gauche conformers and jogs (gtttg) are found for PCs (DMPC: 2.6; DPPC: 2.4), followed by DMPG^– (2.0), phosphatidylethanolamines (DMPE: 1.4; DPPE: 2.0), protonated DMPG (1.5), and phosphatidic acids (DPPA^–: 1.7; DMPA^–: 1.4, DMPA^2–: 1.7). From ²H-NMR measurements of perdeuterated samples of DMPC, DMPA, DPPC, and DPPA the quadrupolar splittings _Qi and the order parameter S _CDi of the CD₂-segments close to the chain ends could be determined whereas splittings in the plateau region of the chains could not be resolved. The quadrupolar splittings are affected by trans-gauche isomerization, long axis rotation, and restricted wobbling motions of the acyl chains. In the simplest assumption, the order parameter S_CD can be expressed as a product of a segmental order parameter S and a lhain order parameter S . For comparison of the different lipids we used average order parameters S_CD, obtained by averaging over all values, and S determined from the total number of gauche conformers per chain by FT-IR-spectroscopy, to calculate an empirical average chain order parameter S. The combination of ²H-NMR and FT-IR results allows the estimation of the relative extent of chain wobbling for the different lipid molecules. S is lowest for PCs (S 0.475) while PEs (S 0.51) and PAs (S0.52) show less chain wobbling.Abbreviations FT-IR Fourier transform infrared - ²H-NMR deuterium nuclear magnetic resonance - DMPC(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylcholine - DMPE(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylethanolamine - DMPA(–d₅₄) (perdeuterated) dimyristoyl-phosphatidic acid - DMPG dimyristoyl-phosphatidylglycerol - DPPC(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylcholine - DPPE(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylethanolamine - DPPA(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidic acid - gtg gauche ^±-trans-gauche^± - gtg gauche^±-trans-gauche^± - dg double gauche - eg end gauche Correspondence to: A. Blume