Stability of the plasma membrane in Saccharomyces cerevisiae enriched with phosphatidylcholine or phosphatidylethanolamine.

Spheroplasts from Saccharomyces cerevisiae NCYC 366, enriched in phosphatidylethanolamine after growth in medium supplemented with 1 mM ethanolamine, were more resistant to osmotic lysis than were spheroplasts from cells grown in the presence of 1 mM choline and enriched in phosphatidylcholine.     

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non-bilayer configurations in liposomes containing phosphatidylethanolamine, thereby membrane leakage.     

The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2(-/-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers.     

Structural differences among phosphatidylcholine, phosphatidylethanolamine, and mixed phosphatidylcholine/phosphatidylethanolamine multilayers: an infrared absorption study

We have examined the infrared absorption spectra from 4000 to 250 cm^?1 of multilayers of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine/phosphatidylethanolamine (1:1 m/m) as a function of hydration, pH, and fatty acid composition. Characteristic splittings of the CH₂ bending and rocking modes and the position of the phosphoryl absorption at ca. 1240 cm^?1 reveal differences in acyl chain packing and head group conformation in the various films. Spectra demonstrate the importance of NH → O hydrogen bonding of the ethanolamine head group and the prerequisite head group conformation (tangent to the multilayer plane) in establishing these structural differences. The general appearance of the P-O-C stretching region (~1050 cm^?1) in the pure and mixed films further supports these conclusions and shows that the spectra clearly distinguish among the different head group orientations. Self-association of phosphatidylethanolamine is sometimes sufficient to prevent formation of mixed phases with phosphatidylcholine at neutral pH. The amount of fine structure, particularly in the low-frequency (800?200 cm^?1) region, in spectra of films of anhydrous, saturated-chain phospholipids decreases considerably when the films are monohydrated, when mixed phases exist, or when there are unsaturations in the acyl chains. These changes likely result from decreased crystal field effects in the spectra as the phosphatide packing density is decreased by any of the above procedures. Furthermore, the absence of other changes upon complete hydration of phosphatidylcholine films suggests that only the initial water is tightly bound to the lipid.     

Ha-ras-transformation alters the metabolism of phosphatidylethanolamine and phosphatidylcholine in NIH 3T3 fibroblasts

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha-ras transformation. All phospholipid fractions were reduced in ras-transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras-cells. Acyl-CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine-cytidylyltransferase and CTP:phosphoethanolamine-chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline- and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP-choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP-ethanolamine pathway.     

Permeability of phosphatidylcholine and phosphatidylethanolamine bilayers

Sodium and glucose effluxes were measured in liposomes formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Vesicles composed of a saturated PC display a local permeability maximum in the region of the lipid transition temperature. The height of this maximum is predominantly a function of the thickness of the hydrocarbon chain region. Liposomes formed from a saturated PE do not display such a permeability maximum and in these vesicles the permeability process appears to be controlled by the head group region. It is postulated that the control exerted by the ethanolamine group is due to the reorganization of water structure it induces at the bilayer surface.     

Evidence for superlattice arrangements in fluid phosphatidylcholine/phosphatidylethanolamine bilayers.

Recently, evidence for cholesterol and phosphatidylcholine (PC) molecules to adapt superlattice arrangements in fluid lipid bilayers has been presented. Whether superlattice arrangements exist in other biologically relevant lipid membranes, such as phosphatidylethanolamine (PE)/PC, is still speculative. In this study, we have examined the physical properties of fluid 1-palmitoyl-2-oleoyl-PC (POPC) and 1-palmitoyl-2-oleoyl-PE (POPE) binary mixtures as a function of the POPE mole fraction (X(PE)) using fluorescence and Fourier transform infrared spectroscopy. At 30 degrees C, i.e., above the Tm of POPE and POPC, deviations, or dips, as well as local data scattering in the excimer-to-monomer fluorescence intensity ratio of intramolecular excimer forming dipyrenylphosphatidylcholine probe in POPE/POPC mixtures were detected at X(PE) approximately 0.04, 0.11, 0.16, 0.26, 0.33, 0.51, 0.66, 0.75, 0.82, 0.91, and 0.94. The above critical values of X(PE) coincide (within +/-0.03) with the critical mole fractions X(HX,PE) or X(R,PE) predicted by a headgroup superlattice model, which assumes that the lipid headgroups form hexagonal or rectangular superlattice, respectively, in the bilayer. Other spectroscopic data, generalized polarization of Laurdan and infrared carbonyl and phosphate stretching frequency, were also collected. Similar agreements between some of the observed critical values of X(PE) from these data and the X(HX,PE) or X(R,PE) values were also found. However, all techniques yielded critical values of X(PE) (e.g., 0.42 and 0.58) that cannot be explained by the present headgroup superlattice model. The effective cross-sectional area of the PE headgroup is smaller than that of the acyl chains. Hence, the relief of "packing frustration" of PE in the presence of PC (larger headgroup than PE) may be one of the major mechanisms in driving the PE and PC components to superlattice-like lateral distributions in the bilayer. We propose that headgroup superlattices may play a significant role in the regulation of membrane lipid compositions in cells.     

Fluorescence studies of dehydroergosterol in phosphatidylethanolamine/phosphatidylcholine bilayers

Our previous fluorescence study has provided indirect evidence that lipid headgroup components tend to adopt regular, superlattice-like lateral distribution in fluid phosphatidylethanolamine/phosphatidylcholine (PE/PC) bilayers (, Biophys. J. 73:1967-1976). Here we have further studied this intriguing phenomenon by making use of the fluorescence properties of a sterol probe, dehydroergosterol (DHE). Fluorescence emission spectra, fluorescence anisotropy (r), and time-resolved fluorescence intensity decays of DHE in 1-palmitoyl-2-oleoyl-PC (POPC)/1-palmitoyl-2-oleoyl-PE (POPE) mixtures were measured as a function of POPE mole fraction (X(PE)) at 23 degrees C. Deviations, including dips or kinks, in the ratio of fluorescence peak intensity at 375 nm/fluorescence peak intensity at 390 nm (I(375)/I(390)), fluorescence decay lifetime (tau), or rotational correlation time (rho) of DHE versus PE composition plots were found at X(PE) approximately 0.10, 0.25, 0.33, 0.65, 0.75, and 0.88. The critical values at X(PE) approximately 0.33 and 0.65 were consistently observed for all measured parameters. In addition, the locations, but not the depth, of the dips for X(PE) < 0.50 did not vary significantly over 10 days of annealing at 23 degrees C. The observed critical values of X(PE) coincide (within +/-0.03) with some of the critical mole fractions predicted by a headgroup superlattice model proposing that the PE and PC headgroups tend to be regularly distributed in the plane of the bilayer. These results agree favorably with those obtained in our previous fluorescence study using dipyrenylPC and Laurdan probes and thus support the proposition that 1) regular arrangement within a domain exists in fluid PE/PC bilayers, and 2) superlattice formation may play a significant role in controlling the lipid composition of cellular membranes (, Proc. Natl. Acad. Sci. USA. 95:4964-4969). The present data provide new information on the physical properties of such superlattice domains, i.e., the dielectric environment and rotational motion of membrane sterols appear to change abruptly as the lipid headgroups exhibit regular superlattice-like distributions in fluid bilayers.     

NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.     

Solid- and liquid-phase equilibria in phosphatidylcholine/phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5 degrees C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mole fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847-854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Solid- and liquid-phase equilibria in phosphatidylcholine / phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5°C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mode fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847–854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Growth rate of myelin figures for phosphatidylcholine and phosphatidylethanolamine.

The growth length of myelin figures (or myelin tubes) was measured for several kinds of phospholipids using optical microscopy. The measurements were done for myelin figures with various thickness of tube wall. In spite of remarkable differences in morphology between the myelin figures of phosphatidylcholine and those of phosphatidylethanolamine, the growth rates for both were adapted to the expression proposed previously. The initial rate and the damping factor of the growth were inversely proportional to the wall thickness of myelin tubes.     

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.     

Effect of propylthiouracil-induced hypothyroidism on phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin synthesis in chick liver microsomes.

Biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin was studied in liver endoplasmic reticulum obtained from newly hatched chicks which were made hypothyroid by feeding 0.2% propylthiouracil. In vitro measurements were made of the specific activities of phosphorylcholine-glyceride (cholinephosphotransferase (EC 2.7.8.2), hosphorylethanolamine-glyceride (ethanolamine-phosphotransferase (EC 2.7.8.1)), and phosphorylcholine-ceramide (ceramide cholinephosphotransferase (EC 2.7.8.3)) transferases in control and hypothyroid chick liver for a period of 40 days. The specific activity of all three transferases began to decline after the chicks were on the propylthiouracil-containing diet for 5 days and steadily declined, reaching levels 10-15% of the controls after 15 days. These low levels were maintained for as long as the chicks were on this diet. Administration of L-thyroxine (15 mug/100 g of body weight) to the hypothyroid chicks caused a marked increase in the specific activities of all three transferases, reaching levels similar to those seen in the control chicks in 36-48 h. The specific activities then declined as the chicks were maintained on the diet of propylthiouracil, reaching the former low levels after 120 h. Administration of cycloheximide alone to the hypothyroid chicks caused a rise in the specific activities of the transferases after 24 h approximately equal to that caused by thyroxine alone, while thyroxine and cycloheximide together were no different than either alone. These studies indicate that in some manner circulating thyroxine controls the activities of enzymes involved in the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin in chick liver endoplasmic reticulum. There was no evidence that induction of hypothyroidism by propylthiouracil had any effect on the activities of these enzymes in the CNS.     

Effect of bile salts on monolayer curvature of a phosphatidylethanolamine/water model membrane system.

A partial phase diagram of the ternary system dioleoylphosphatidylethanolamine (DOPE)/sodium cholate/water has been determined using 31P Nuclear Magnetic Resonance (NMR) spectroscopy. In the absence of cholate, it is well known that the DOPE/water system forms a reversed hexagonal (HII) phase. We have found that addition of even small amounts of cholate to the DOPE/water system leads to a transition to a lamellar (L alpha) phase. At higher cholate concentrations, a cubic (I) phase (low water content) or a micellar solution (L1) phase (high water content) is present. Thus, cholate molecules have a strong tendency to alter the lipid monolayer curvature. Increasing the concentration of cholate changes the curvature of DOPE from negative (HII phase), through zero (L alpha phase), and finally to a phase of positive curvature (micellar solution). This observation can be rationalized in terms of the molecular structure of cholate, which is amphipathic and has one hydrophobic and one hydrophilic side of the steroid ring system. The cholate molecules have a tendency to lie flat on the lipid aggregate surface, thereby increasing the effective interfacial area of the polar head groups, and altering the curvature free energy of the system.     

Mass spectrometry analysis of oxidized phosphatidylcholine and phosphatidylethanolamine

Oxidized phospholipids (OxPLs) are rapidly becoming recognized as important mediators of cellular and immune signaling. They are generated either enzymatically or non-enzymatically and 100s of structures exist of which only a small fraction have been analyzed to date. Pleiotropic activities, including regulation of adhesion molecule expression, pro-coagulant activity and inhibition of Toll-like receptor signaling have been observed and some are detected in models of human and animal disease, including atherosclerosis and infection. More recently, the acute generation of specific oxidized phospholipids by cellular enzymes in immune cells was reported. Assays for analysis and quantification of OxPLs were first developed approx 15years ago, primarily for hydro(pero)xy-species. Many were based on monitoring a single precursor ion with/without LC separation, based on the PL headgroup. Others combined LC with monitoring precursor to product transitions, but were unable to provide information regarding position of oxidation on unsaturated sn-2 fatty acid due to sensitivity issues. More recently, LC/MS/MS methods for specific OxPLs have been reported that enable high sensitivity quantitation in biological samples. In this review, widely used methods for detecting and quantifying various classes of OxPL will be summarized, along with practical advice for their use. In particular, the focus will be on LC/MS/MS, which today is almost universally the method of choice.     

Bilayer to non-bilayer transition in mixtures of phosphatidylethanolamine and phosphatidylcholine: Implications for membrane properties

The structural phases in various mixtures of soybean phosphatidylethanolamine and egg phosphatidylcholine were studied by X-ray diffraction, freeze fracture electron microscopy, and ³¹P NMR. An intermediate state between bilayer and hexagonal structures was found at a composition of 10–25 mol% of phosphatidylcholine. The intermediate state consists of closely packed multilayers, together with arrays of lipidic intramembranous particles. The arrays of lipidic intramembranous particles, possibly membrane invaginations, give rise to an anisotropic ³¹P NMR spectrum commonly accredited to a hexagonal structure. A phase diagram of this mixed system is proposed. The compositional range at which the intermediate state exists coincides with the range of maximal mitochondrial ATPase activity when these lipids are used in reconstitution experiments.     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.