Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of diacylglycerols on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2.

The effects of a series of diacylglycerols (DAGs) with varying acyl chain lengths and degree of unsaturation on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2 (PL-A2S) were studied using two lipid substrates: dipalmitoylphosphatidylcholine (DPPC) or bovine liver phosphatidylcholine (BL-PC). The activities of the phospholipases critically depended on the chain length and degree of unsaturation of the added DAGs and on the chemical composition of the substrate. The effects of DAGs on cobra or bee venom PL-A2S were similar, but significantly different from the pig pancreatic PL-A2. The data, taken together with our previous NMR studies on physicochemical effects of these DAGs on lipid bilayer structure [De Boeck, H., & Zidovetzki, R. (1989) Biochemistry 28, 7439; (1992) Biochemistry 31, 623], allowed detailed correlation of the type of a bilayer perturbation induced by DAG with the activation or inhibition of the phospholipase on the same system. In general, the activation of the phospholipases correlated with the DAG-induced defects of the lipid bilayer structure. The results, however, argue against general designation of DAGs as "activators" or "inhibitors" of PL-A2S. Thus, for example, diolein activated phospholipases with the BL-PC lipid substrate, but inhibited them with the DPPC substrate. Dihexanoylglycerol and dioctanoylglycerol inhibited pig pancreatic PL-A2 with both lipid substrates and inhibited cobra or been venom PL-A2 with the DPPC substrate, but activated the latter two enzymes with the BL-PC substrate. Longer-chain DAGs (C greater than 12), which induce lateral phase separation of the bilayers into the regions of different fluidities, activated all PL-A2S with both lipid substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.     

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Effects of dipalmitoylglycerol and fatty acids on membrane structure and protein kinase C activity.

The individual and combined effects of the saturated diacylglycerol (DAG) dipalmitin (DP) and saturated or polyunsaturated unesterified fatty acids (PUFAs) on both the structure of phosphatidylcholine/phosphatidylserine (PC/PS; 4:1 mol/mol) bilayers and on protein kinase C (PKC) activity were studied using 2H nuclear magnetic resonance (NMR) and enzyme activity assays. In the absence of DP, PUFAs only slightly activated PKC whereas palmitic acid had no effect. In the absence of fatty acids, DP induced lateral phase separation of the bilayer into liquid-crystalline and gel phases. Under these conditions virtually all DP was sequestered into the gel phase and no activation of PKC was observed. The addition of polyunsaturated arachidonic or docosahexaenoic acids to the DP-containing bilayers significantly increased the relative amounts of DP and other lipid components in the liquid-crystalline phase, correlating with a dramatic increase in PKC activity. Furthermore, the effect was greater with PS, resulting in an enrichment of PS in the liquid-crystalline domains. In the presence of DP, palmitic acid did not decrease the amount of gel phase lipid and had no effect on PKC activity. The results explain the observed lack of PKC-activating capacity of long-chain saturated DAGs as due to the sequestration of DAG into gel domains wherein it is complexed with phospholipids and thus not available for the required interaction with the enzyme.     

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry.

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.     

Determination of the molecular species composition of diacylglycerols in human adipose tissue by solid-phase extraction and gas chromatography on a polar phase

The free diacylglycerols (DAGs) in adipose tissue are involved in the metabolism of stored lipids and hence are related to the supply of fatty acids for other tissues. This paper describes a simple, fast, and reproducible method for the identification and quantification of different molecular species of DAGs in human adipose tissue. The method comprised solid-phase extraction on a diol-bonded phase column combined with capillary GC analysis of silylated DAG derivatives on a polar phase (65% phenylmethylsilicone). Separation of the DAGs was achieved based on chain length, isomeric structure (1,2- and 1,3-DAGs), and degree of unsaturation. The main DAGs were 1,2-OO, 1,2-OP, 1,2-LO and 1,2-LP. The composition was corroborated by analysis of the component fatty acids of the DAGs, 18:1(n-9), 16:0, and 18:2(n-6) being the three major fatty acids obtained.     

Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study

We present a combined theoretical (molecular dynamics, MD) and experimental (differential scanning calorimetry, DSC) study of the effect of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl chain-labeled fluorescent phospholipid analogs (C6-NBD-PC and C12-NBD-PC) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. DSC measurements reveal that < 1 mol% of NBD-PC causes elimination of the pre-transition and a large loss of cooperativity of the main transition of DPPC. Labeling with C6-NBD-PC or C12-NBD-PC shifts the main transition temperature to lower or higher values, respectively. Following our recent report on the location and dynamics of these probes (BBA 1768 (2007) 467-478) in fluid phase DPPC, we present a detailed analysis of 100-ns MD simulations of systems containing either C6-NBD-PC or C12-NBD-PC, focused on their influence on several properties of the host bilayer. Whereas most monitored parameters are not severely affected for 1.6 mol% of probe, for the higher concentration studied (6.2 mol%) important differences are evident. In agreement with published reports, we observed that the average area per phospholipid molecule increases, whereas DPPC acyl chain order parameters decrease. Moreover, we predict that incorporation of NBD-PC should increase the electrostatic potential across the bilayer and, especially for C12-NBD-PC, slow lateral diffusion of DPPC molecules and rotational mobility of DPPC acyl chains.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane alpha-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.     

A molecular basis explanation of the dynamic and thermal effects of vinblastine sulfate upon dipalmitoylphosphatidylcholine bilayer membranes

Differential scanning calorimetry has been employed to study the thermal effects of vinblastine sulfate upon aqueous, single and multiple bilayer dispersions of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC). The calorimetric results summarized to an increase in the gel to liquid-crystalline phase transition enthalpy and the abolishment of the L(beta)' (gel phase) to P(beta)' (ripple phase) pretransition for the uni- and multilamellar dispersions, as well as an increase in the transition temperature T(m) and the transition cooperativity for single bilayer DPPC/vinblastine mixed vesicles, are consistent with an induced, partially interdigitated, gel phase. Computational analysis has been successfully applied to clarify the intermolecular effects and verify the feasibility of the proposed interdigitation for the vinblastine sulfate molecules and also for the ursodeoxycholic acid (UDCAH) and bromocylated taxanes, which have been shown to induce an interdigitated gel phase in DPPC bilayers.     

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane α-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic alignment and orientational order of dipalmitoylphosphatidylcholine bilayers containing palmitoyllysophosphatidylcholine

Mixed bilayers of 1-palmitoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine; PaLPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine; DPPC) have been investigated by 2H-NMR and 31P-NMR spectroscopy. Binary phospholipid mixtures were studied in which the acyl chains of one or the other component were perdeuterated. At temperatures below the main order-disorder phase transition, the mixed PaLPC/DPPC bilayers appear to coexist with PaLPC micelles. The micelles disappear at temperatures above the phase transition, where mixed bilayers in the liquid-crystalline state are formed. The orientational order of the alkyl chains of the PaLPC component is essentially identical to that of the DPPC component in the mixed bilayers, both in the low temperature and liquid-crystalline phases. However, the presence of PaLPC perturbs the segmental ordering of DPPC as compared to the pure system. The order is increased in the low-temperature phase, where effective diffusion of the chains about their long axes occurs, but is decreased in the liquid-crystalline phase compared to pure DPPC bilayers. The mixed liquid-crystalline bilayers orient preferentially with their director axes perpendicular to the magnetic field. This alignment is easily observed in 31P- and 2H-NMR spectra, where the intensity of the perpendicular edges of the lineshapes is pronounced. One possible explanation of the magnetic alignment involves alteration of the curvature free energy of the DPPC bilayer due to incorporation of PaLPC in the mixed membranes.     

Globoside with spin-labelled fatty acid: bilayer lateral distribution and immune recognition

We have critically addressed the question of lateral distribution of glycolipids in bilayer membranes, and the effect of glycolipid fatty acid chain length upon such distribution. For this purpose we synthesised the complex neutral glycosphingolipid, globoside, with spin-labelled fatty acid. Base hydrolysis to remove the natural fatty acid was found to deacetylate the GalNAc residue concomitantly, necessitating application of the synthetic route described for gangliosides by Neuenhofer et al. (Biochemistry 24, 525-532 (1985)). Globosides were produced with 18-carbon and 24-carbon fatty acids bearing a spin label at the C-16 position. Spin-labelled globosides were incorporated at 2 and 10 mol% into rigid, highly cooperative bilayer matrices of 1,2-dipalmitoylglycerophosphocholine (DPPC) and also into semi-fluid, non-cooperative membranes of DPPC/cholesterol. Recorded electron paramagnetic resonance (EPR) spectra were analysed by comparison with a library of standards representing samples of known composition. Spectra were manipulated using a computer program which permitted linear combination of standards to stimulate coexistence of laterally separated domains of different composition. The most important conclusions were as follows: (1) at least 80% of the globoside was definitely not confined to domains highly enriched in glycolipid, although there was evidence of binary-phase separation in the rigid DPPC/globoside matrix; (2) the presence of 33 mol% cholesterol reduced the evidence of globoside phase separation; (3) there was remarkably little difference in results whether the globoside fatty acid chain length was similar to that of the phospholipid host matrix or eight carbons longer. Temperature profiles derived over the phase-transition region of DPPC using spin-labelled globoside or an unattached amphiphilic spin label were consistent with these findings. The same systems lent themselves to consideration of the role of glycolipid fatty acid chan length and cholesterol in determining glycolipid crypticity in membranes: (1) polyclonal anti-globoside IgG bound to globoside in DPPC liposomes without inducing agglutination. (2) The same antibodies did agglutinate DPPC/cholesterol liposomes bearing globoside. (3) The effect of cholesterol probably was upon glycolipid dynamics or attitude in the membrane, rather than upon distribution. (4) These observations were basically unaffected by the choice of 18-carbon vs. 24-carbon glycolipid fatty acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

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.