Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.     

Interaction of salt with ether- and ester-linked phospholipid bilayers

A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. Experiments show that this chemical difference in headgroup-tail linkage does produce distinct differences in model bilayer properties. Here we examine the effects of salt on bilayer structure in the case of an ether-linked lipid bilayer. We use molecular dynamics simulations and compare equilibrium properties of two model lipid bilayers in NaCl salt solution – POPC and its ether-linked analog that we refer to as HOPC. We make the following key observations. The headgroup region of HOPC “adsorbs” fewer ions compared to the headgroup region of POPC. Consistent with this, we note that the Debye screening length in the HOPC system is ∼ 10% shorter than that in the POPC system. Herein, we introduce a protocol to identify the lipid-water interfacial boundary that reproduces the bulk salt distribution consistent with Gouy-Chapman theory. We also note that the HOPC bilayer has excess solvent in the headgroup region when compared to POPC, coinciding with a trough in the electrostatic potential. Waters in this region have longer autocorrelation times and smaller lateral diffusion rates compared to the corresponding region in the POPC bilayer, suggesting that the waters in HOPC are more strongly coordinated to the lipid headgroups. Furthermore, we note that it is this region of tightly coordinated waters in the HOPC system that has a lower density of Na⁺ ions. Based on these observations we conclude that an ether-linked lipid bilayer has a lower binding affinity for Na⁺ compared to an ester-linked lipid bilayer.     

Deuterium NMR investigation of ether- and ester-linked phosphatidylcholine bilayers

Deuterium nuclear magnetic resonance (2H NMR) spectra of specifically head-group- and chain-deuterated ester- and ether-linked phosphatidylcholine bilayers were studied as a function of temperature over the range -33 to 50 degrees C. Head-group-deuterated dihexadecylphosphatidylcholine ([alpha-2H2]DHPC) bilayers yield line shapes and spin-lattice relaxation times similar to those observed for its ester-linked counterpart, dipalmitoylphosphatidylcholine ([alpha-2H2]DPPC), in the high-temperature ripple and L alpha bilayer phases. These results indicate the ether linkage has no effect on the dynamics or the orientational order at the alpha-C2H2 segment of the phosphocholine head group. At all temperatures, the 2H NMR spectra of chain-deuterated 1,2[1',1'-2H2]DHPC bilayers exhibit a reduced spectral width compared to 1,2[2',2'-2H2]DPPC bilayers. The most significant feature of the deuterated alkyl chain spectrum of DHPC at 45 degrees C is the observation of four separate quadrupolar splittings from the alpha-methylene segments of the alkyl chains, in comparison to the three quadrupolar splittings reported previously from the alpha-methylene segments of the acyl chains of DPPC. Spin-lattice relaxation experiments performed on DHPC suggest an assignment of the two smaller and the two larger quadrupolar splittings to separate alkyl chains, respectively. Low-temperature (T less than or equal to -20 degrees C) gel-phase spectra of deuterated head-group [alpha-2H2]DHPC remain an order of magnitude narrower than those observed for [alpha-2H2]DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-pressure infrared spectroscopy of ether- and ester-linked phosphatidylcholine aqueous dispersions.

Infrared spectra of aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and its ether-linked analogue, 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC), were measured in a diamond anvil cell at 28 degrees C as a function of pressure up to 20 kbar. Although these two lipids differ only in the linkages to the saturated hydrocarbon chains, significant differences were observed in their barotropic behavior. Most notable were the magnitudes of the pressure-induced correlation field splittings of the methylene scissoring and rocking modes, and the relative intensities of the corresponding component bands. In the case of the scissoring mode, not only can the correlation field component band be resolved at a lower pressure in DHPC (1.2 kbar, as compared with 2.2 kbar in DPPC), but the initial magnitude of the correlation field splitting in DHPC, particularly less than 9 kbar, is significantly greater than that observed in DPPC. These differences are attributed to the presence of an interdigitated lamellar gel phase in DHPC. At all pressures where the correlation field component band delta'CH2 can be resolved, the relative peak height/intensity ratio R = I delta'/I delta is greater in DPPC than in DHPC, suggesting that this parameter may be useful as a test of interdigitation.     

Comparative study of the gel phases of ether- and ester-linked phosphatidylcholines

Calorimetric, X-ray diffraction, and 31P nuclear magnetic resonance (NMR) studies of aqueous dispersions of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) gel phases at low temperatures (-60 to 22 degrees C) show thermal, structural, and dynamic differences when compared to aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) gel phases at corresponding temperatures. Differential scanning calorimetry of DHPC dispersions demonstrates a reversible, low-enthalpy "subtransition" at 4 degrees C in contrast to the conditionally reversible, high-enthalpy subtransition observed at 17 degrees C for annealed DPPC bilayers. X-ray diffraction studies indicate that DHPC dispersions form a lamellar gel phase with dav congruent to 46 A both above and below the "subtransition". It is suggested that the reduced dav observed for DHPC (46 A as compared to 64 A in DPPC) is due to an interdigitated lamellar gel phase which exists at all temperatures below the pretransition at 35 degrees C. 31P NMR spectra of DHPC gel-phase bilayers show an axially symmetric chemical shift anisotropy powder pattern which remains sharp down to -20 degrees C, suggesting the presence of fast axial diffusion. In contrast, 31P spectra of DPPC bilayers indicate this type of motion is frozen out at approximately 0 degrees C.     

Highly sensitive determination and characterization of intact cellular ester-linked phospholipids using liquid chromatography-plasma spray mass spectrometry

Liquid chromatographic class separations of common cellular phospholipids combined with plasma spray ionization of the effluents were investigated. Comparison with true thermospray ionization involving ammonium acetate buffering revealed a gain in total ionization in the plasma spray of a factor of approximately 10 using a cation-exchange column and a solvent mixture consisting of acetonitrile-methanol-water (400:100:15, v/v). Plasma spray ionization studies of bovine brain polyphosphoinositides interrelated by the phosphate content in the inositol moeity showed almost identical monoglyceride and diglyceride ion clusters, indicating possibilities of studying the biochemical turnover of such phospholipids. Plasma spray ionization liquid chromatography-mass spectrometry of bacterial membrane phospholipids (Pseudomonas fluorescens) revealed possibilities of obtaining indications of individual fatty acid compositions from the spectra of the phosphatidylinositol and phosphatidylethanolamine fractions present. Conventional gas chromatographic fatty acid analysis agreed with the direct mass spectrometric structure elucidations. Interestingly, the two phospholipid classes had different relative fatty acid compositions with a significantly higher degree of cyclic fatty acids in the phosphatidyl ethanolamines. Plasma spray ionization yielded linear dose-response curves for both the monoglyceride and diglyceride fragment signals in the selected-ion monitoring mode. The detection limit for the monoglyceride and diglyceride species of phosphatidylcholine under the chromatographic and mass spectrometric conditions used was found to be in the picogram range.     

Distribution of omega-3 and omega-6 fatty acids in the ether- and ester-linked phosphoglycerides from tissues of the rainbow trout, Salmo gairdneri

1. Data presented here demonstrate that polyunsaturated fatty acids in the phospholipids of rainbow trout tissues are compartmentalized differently than in mammalian tissues. 2. We have determined the distribution of omega-3 (n-3) and omega-6 (n-6) fatty acids in the alkyl-, alk-1-enyl-, and diacyl- subclasses of phosphatidylcholines (PC), phosphatidyl-ethanolamines (PE), phosphatidylinositols (PI), and phosphatidylserines (PS) from gill, kidney and spleen of rainbow trout. 3. Alkyl-linked PC and alk-1-enyl-linked PE were the most abundant ether-containing phospholipids, amounting to 10-15% of each class; no ether-linked PI or PS was detected. 4. C20:4 n-6 was found in high concentrations only in PI; the n-3 fatty acids were found in highest concentration in the ether-linked phospholipids as compared with the diacyl subclasses and C20:5 n-3 was especially prevalent in 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and C22:6 n-3 was prevalent in PS.     

Angiotensin and bradykinin interactions with phospholipids

Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp¹)-angiotensins. The apparent dissociation constant of this complex in chloroform was 10^?5 M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behavior of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp¹)-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo.     

Bilayer lipid membranes (BLM): Study of antigen-antibody interactions

Previous work of del Castillo and co-workers has shown that bilayer lipid membranes (BLM) can be used as transducers for detection of antigen-antibody reactions. The present experiments extend the previous work by incorporating complement into the BLM system. The results indicate that the antigen-antibody complex or the complement has no ability to affect the BLM system separately, but when carefully combined they will destabilize the BLM even at a much reduced concentration. Further development using the BLM as a tool for investigating immunological reactions is suggested.     

Angiotensin and bradykinin interactions with phospholipids.

Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp1)-angiotensins. The apparent dissociation constant of this complex in chloroform was 10(-5) M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behavior of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp1)-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo.     

Calcium- and calmodulin-sensitive interactions of calcineurin with phospholipids

Physical association of calcineurin with phosphatidylserine (PS) or phosphatidylglycerol (PG) was observed by molecular exclusion chromatography; the enzyme did not associate with phosphatidylethanolamine or phosphatidylcholine. The interactions with PS and PG were enhanced by Ca2+ which implicates a regulatory role for the Ca2+-binding subunit in this process. Addition of PG or PS to standard calcineurin assays elicited profound changes in enzymatic activity; phosphatidylcholine and phosphatidylethanolamine were without effect. Up to 23-fold stimulation of the calmodulin-independent activity was observed with phosphorylated histone H1 or synapsin I as the substrates. In contrast, the activity toward p-nitrophenyl phosphate and tyrosine phosphate was found to be inhibited. A characterization and comparison of the two opposite responses showed that: the phospholipids had insignificant effects on the Km for substrates, the phospholipid specificity for activation and inhibition was nearly indistinguishable, half-maximal activation and inhibition were obtained at similar concentrations of PG (K0.5 = 0.21 and 0.14 mg/ml, respectively), and calmodulin enhanced the responses to PG (K0.5 = 0.064 and 0.033 mg/ml for activation and inhibition, respectively) to similar extents. Together, these observations demonstrate that the two substrate-dependent responses of calcineurin are due to the association of the phosphatase with phospholipids and not a result of substrate-phospholipid interactions. This suggests that Ca2+- and calmodulin-stimulated interactions of calcineurin with acidic phospholipids may play a role in regulating the substrate specificity of this multifunctional phosphatase.     

Lipid dependence of peptide-membrane interactions: Bilayer affinity and aggregation of the peptide alamethicin

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid-peptide interactions.     

Investigation on the interactions between pirarubicin and phospholipids

Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy and quantum calculation based on molecular modeling were applied to investigate the interaction between pirarubicin (THP), an anthracycline antibiotic frequently used in chemotherapy, and zwitterionic distearoylphosphatidylcholine (DSPC) or anionic distearoylphosphatidylglycerol (DSPG). DSC and FTIR studies suggested that DSPG bilayers were less perturbed by THP than those of DSPC, and this might be due to the strong interactions between NH₃⁺ of THP and the phosphate (PO₂⁻) group in the polar head of DSPG, which limit the further access of THP into its bilayers. Quantum calculation results based on molecular modeling could further confirm the DSC and FTIR conclusions. Meanwhile, it could well translate the calorimetric and spectroscopic phenomena into the underlying physical knowledge. Interactions between THP and phospholipids can play a critical role in the liposomal drug delivery system, especially in the safety mechanism elucidation and rational formulation design.     

Non-polar interactions between cholesterol and phospholipids: a molecular dynamics simulation study

A 15-ns molecular dynamics simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer containing approximately 22 mol% Chol was carried out. An 8-ns trajectory was analysed to investigate the effect of Chol on the chain packing in the bilayer core. While the packing of DMPC chains on the smooth alpha-face side of the Chol ring is similar to that in the pure DMPC bilayer, the packing on the rough beta-face side is less regular and less tight. Two methyl groups located on the Chol beta-face disturb the packing; in effect, van der Waals (vdW) interactions between Chol rings and DMPC chains are weaker than the ones between sole DMPC chains. VdW interactions between an alkyl chain of DMPC and an isooctyl tail of Chol are similarly strong as those between two DMPC chains.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.     

Membrane interactions of tetanus and botulinum neurotoxins: a photolabelling study with photoactivatable phospholipids

Tetanus and botulinum neurotoxins (TeNT and BoNT) bind strongly and specifically to the nervous tissue, as it can be inferred from their potency and from their effects restricted to the nervous system. The molecular basis of these properties are presently unknown. As a first approach, we have investigated the interaction of TeNT and BoNT with model membranes by photolabelling with phospholipid analogues carrying the photoreceptor group at different positions of the lipid molecule in order to probe different membrane regions. We found that at neutral pH TeNT and BoNTs (type A, B and E) adsorb onto the surface of negatively charged liposomes. Polysialogangliosides increase this interaction only slightly thus suggesting that they provide a minor contribution to toxin lipid binding. On this basis we propose that clostridial neurotoxins bind to lipids via both a predominant unspecific interaction with negatively charged lipids (including gangliosides) and a specific, but weaker, interaction with polysialogangliosides. At acidic pH values both chains of these neurotoxins are labelled strongly by photogroups located in the hydrophobic milieu of the membrane with a pH dependence that overlaps the range of pH values reached in the endosomal lumen. This result is consistent with their insertion into the lipid bilayer in agreement with the idea that clostridial neurotoxins may penetrate into cells via intracellular low pH compartments.     

Anionic phospholipids inhibit apolipoprotein E--low-density lipoprotein receptor interactions

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity.     

Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: human erythrocyte glycophorin and phospholipids

Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The 31P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.