Comparative physical studies of phosphatidylsulfocholine and phosphatidylcholine. Calorimetry,fluorescence polarization and electron paramagnetic resonance spectroscopy

Transition temperatures of phosphatidylsulfocholines (PSCs; di-14 : 0-, di-16 : 0-, di-18 : 0- and di-18 : 1-) were compared with those of the corresponding phosphatidylcholines (PCs) using the techniques of differential scanning calorimetry, fluorescent polarization with diphenylhexatriene (DPH) and cis- and trans- parinaric acids as probes, and electron paramagnetic resonance (EPR) with 5-doxyl stearic acid as probe. Liposomal dispersions of the sulfonium analogs showed the typical multibilayer structure by electron microscopy (EM) and were in general very similar in physical behaviour to those of the corresponding PCs. However, the fully hydrated saturated PSCs consistently showed sharp main transitions 2–4°C above those of the corresponding PCs, by all three techniques used; the unsaturated PSC (di-18 : 1) had a transition 2–3°C below that of di-18 : 1-PC and only the di-14 : 0-PSC and di-18 : 1-PSC showed a well-defined pretransition. Fluorescence polarization studies with cis- and trans-parinaric acids showed that the PSC bilayers were less ordered than the corresponding PC bilayers in both the gel and liquid crystalline states.These results provide a rationale for the observed ability of the sulfonium analogs to substitute for PC in some natural membranes.     

Membrane properties of plant sterols in phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization

The increased use of plant sterols as cholesterol-lowering agents warrants further research on the possible effects of plant sterols in membranes. In this study, the effects of the incorporation of cholesterol, campesterol, β-sitosterol and stigmasterol in phospholipid bilayers were investigated by differential scanning calorimetry (DSC), resonance energy transfer (RET) between trans parinaric acid (tPA) and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC), and Triton X-100-induced solubilization. The phospholipids used were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), d-erythro-N-palmitoyl-sphingomyelin (PSM), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In DSC experiments, it was demonstrated that the sterols differed in their effect on the melting temperatures of both the sterol-poor and the sterol-rich domains in DPPC and PSM bilayers. The plant sterols gave rise to lower temperatures of both transitions, when compared with cholesterol. The plant sterols also resulted in lower transition temperatures, in comparison with cholesterol, when sterol-containing DPPC and PSM bilayers were investigated by RET. In the detergent solubilization experiments, the total molar ratio between Triton X-100 and POPC at the onset of solubilization (R_t,sat) was higher for bilayers containing plant sterols, in comparison with membranes containing cholesterol. Taken together, the observations presented in this study indicate that campesterol, β-sitosterol and stigmasterol interacted less favorably than cholesterol with the phospholipids, leading to measurable differences in their domain properties.     

Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (L_c), lamellar gel (L_β′), ripple gel (P_β′) and liquid crystal (L_α), in turn. The L_c phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L_βI) phase appeared above the critical interdigitation pressure (CIP) between the L_β′ and P_β′ phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F″₄₉₇/F″₄₃₀ value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L_β′/L_βI phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L_βI phase in the SPPC bilayer has a less polar “pocket” formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Importance of the Sphingoid Base Length for the Membrane Properties of?Ceramides

The sphingoid bases of sphingolipids, including ceramides, can vary in length from 12 to >20 carbons. To study how such length variation affects the bilayer properties of ceramides, we synthesized ceramides consisting of a C12-, C14-, C16-, C18-, or C20-sphing-4-enin derivative coupled to palmitic acid. The ceramides were studied in mixtures with palmitoyloleoylphosphocholine (POPC) and/or palmitoylsphingomyelin (PSM), and in more complex bilayers also containing cholesterol. The trans-parinaric acid lifetimes showed that 12:1- and 14:1-PCer failed to increase the order of POPC bilayers, whereas 16:1-, 18:1-, and 20:1-PCer induced ordered- or gel-phase formation. Nevertheless, all of the analogs were able to thermally stabilize PSM, and a chain-length-dependent increase in the main phase transition temperature of equimolar PSM/Cer bilayers was revealed by differential scanning calorimetry. Similar thermal stabilization of PSM-rich domains by the ceramides was observed in POPC bilayers with a trans-parinaric acid-quenching assay. A cholestatrienol-quenching assay and sterol partitioning experiments showed that 18:1- and 20:1-PCer formed sterol-excluding gel phases with PSM, reducing the overall bilayer affinity of sterol. The effect of 16:1-PCer on sterol distribution was less dramatic, and no displacement of sterol from the PSM environment was observed with 12:1- and 14:1-PCer. The results are discussed in relation to other structural features that affect the bilayer properties of ceramides.     

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)     

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro

Cytosolic phospholipase A₂ alpha (cPLA₂α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA₂α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA₂α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA₂α. Such promiscuous binding would explain the previous finding that cPLA₂α has both PLA₁ and PLA₂ activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA₂α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA₂α.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid

The activation of H⁺-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H⁺-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H⁺-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.     

Ether- versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains

We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces F_b decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different F_b values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.     

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T_m) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T_m values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T_m-diagrams are shown, from which the effects of the number and the position of one to five cis carbon–carbon double bonds on T_m can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T_m trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon–carbon double bonds as seen in the T_m-diagram are presented.     

Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

Ether- versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains

We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces F_b decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different F_b values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C_n = 12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland,corpus allatum

In the conventional view, the winter adaptation of membrane lipids is induced by temperature decrease. We propose that winter remodelling of membranes in Pyrrhocoris apterus is triggered by short-day photoperiod before the temperature decrease and changes caused by cold temperature represent the later phase of adaptation. The induction of diapause by short-day photoperiod results in an accumulation of phosphatidylethanolamine (PE) molecular species with C_16:0/C_18:2 acyl chains esterified to sn-1/sn-2 positions of glycerol at the expense of C_18:0/C_18:2. Proportions of C_16:0/C_18:2-PE are enhanced in short-day compared to long-day insects in both thoracic muscles (TM, 15.0 vs. 8.2%) and fat bodies (FB, 24.9 vs. 13.6 %). Proportions of C_16:0/C_18:2-PE are further enhanced during cold acclimation (to 26.5% in TM, 33.6 % in FB) at the expense of a more saturated species, C_18:0/C_18:1-PE. These changes are less prominent in phosphatidylcholines (PC). The effect of photoperiod seems to be mediated via the corpus allatum. Long-day non-diapause females deprived of their corpus allatum have the phospholipid molecular species profile similar to that found in short-day diapausing females. While the acyl chain remodelling is regulated by both photoperiod and temperature, the head group composition is regulated by temperature only. Similar to most other organisms, the level of PE is higher (50.3 vs. 43.5% in TM, 44.3 vs. 37.8% in FB) and that of PC is lower (35.9 vs. 40.2% in TM, 41.6 vs. 46.1 % in FB) at cold temperatures (≤1°C) compared to warm temperatures (≥16°C). In contrast to a general rule, the PE is less unsaturated than PC. In both TM and FB, proportions of unsaturated/unsaturated molecular species are consistently high in PC (56.3-67.5% in TM, 59.2-66.6% in FB), while they are consistently low in PE (19.1-26.7% in TM, 12.1-15.1% in FB). An adaptive significance of changes in the phospholipid composition for the low temperature and/or dehydration stress is discussed in relation to known physical properties of phospholipids.