Translational diffusion and fluid domain connectivity in a two-component, two-phase phospholipid bilayer.

The two-dimensional connectivity is examined for mixed bilayers of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) as a function of composition and temperature at constant pressure using the fluorescence recovery after photobleaching (FRAP) method. These phospholipid mixtures exhibit peritectic behavior with a large region in which both gel and liquid crystalline phases coexist. Dilauroyl phosphatidylethanolamine covalently linked through the amino function in its head group to the fluorescent nitrobenzodiazolyl group (NBD-DLPE) was used as the fluorescent probe in this study, because it was found to partition almost exclusively in the liquid crystalline phase. The results of these studies show the line of connectivity to be close to the liquidus line on the phase diagram over a rather broad range of concentrations. In this range, a gel phase comprising approximately 20% of the system disconnects a liquid crystalline phase comprising 80% of the system. The implications of this result are discussed for domain shape and the organization of biological membrane components.     

Ca2+-induced phase separation in phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca2+-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidylethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94--100% at 23 degrees C and 84--88% at 40 degrees C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74--85% at 23 degrees C and 61--79% at 40 degrees C. Ca2+ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was 1.4 . 10(-7) M for phosphatidylserine-phosphatidylethanolamine and 1.2 . 10(-6) M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca2+ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17 degrees C) and phosphatidylcholine (-15 degrees C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca2+-chelated solid-phase phosphatidylserine domain.     

Phospholipid transfer between vesicles. Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

Determination of fluid and gel domain sizes in two-component, two-phase lipid bilayers. An electron spin resonance spin label study.

The average sizes of fluid and gel domains in the two-component, two-phase system formed from mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine were determined from an analysis of the electron spin resonance spectral lineshapes of a dimyristoyl phosphatidylcholine-nitroxide spin label as a function of spin label concentration. The ratio, R, of the intensities measured at two magnetic field strengths was found to be diagnostic of a statistical distribution of spin labels in disconnected domains. R is defined as V'/2Vpp, where Vpp is the maximum intensity and V' is the intensity at a position in the wings of a first derivative electron spin resonance line that is a constant multiple of the peak-to-peak linewidth. The intensity ratio for Gaussian or Voigt lineshapes is less than or equal to the value for a Lorentzian lineshape. The intensity ratio was found to be greater than the value for a Lorentzian line when spectra from disconnected domains containing a statistical distribution of spin labels undergoing spin-spin interactions were summed. The intensity ratio, R, calculated by spectral simulations as a function of the average number of labels per domain, N, was found to increase to a maximum with increasing N and then to decrease. The dependence on spin label concentration of the experimentally measured intensity ratios paralleled this predicted behavior. A method is presented to calculate the average number of lipids per fluid or gel domain based on a knowledge of R, and of the distribution of the spin label between the fluid and gel phases determined from the phase diagram. The results demonstrate that the number of lipids per domain increases linearly from a fixed number of nucleation sites, as the fraction of the phase that is disconnected increases. At any given mole fraction of the particular phase, the gel domains are bigger than the fluid domains because they have a lower nucleation density. The results also suggest that the disconnected domains are, in most cases, nonrandomly distributed in the plane of the bilayer.     

Phospholipid transfer between vesicles: Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicles was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they were transferred to the nonspin-labeled acceptor vesicles. A lowe transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles of a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a ³¹P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

Lipid phase states influence glycophorin reconstitution

Reconstitution of glycophorin into dimyristoyl phosphatidylcholine and sphingomyelin vesicles was sub-maximal below the phase transition temperatures of these lipids. Reconstitution of glycophorin into diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine liposomes was maximal within a range of temperatures below the phase transition temperatures of dimyristoyl phosphatidylcholine and sphingomyelin but above the phase transition temperatures of diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine. These findings indicate a greater tendency for reconstitution of glycophorin into fluid as opposed to solid lipid phases.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

Is the Distribution of α-Tocopherol in Membranes Consistent with Its Putative Functions?

Vitamin E acts as an antioxidant and stabilizer of membranes. Other functions of vitamin E unrelated to its effects on membranes are emerging. Vitamin E partitions into the lipid bilayer matrix of membranes. It orients perpendicularly to the plane of the membrane with the hydroxyl group pointing to the lipid-water interface. The vitamin is not randomly distributed in the plane of the membrane but tends to form clusters. These clusters appear to be composed of vitamin E and phosphatidylcholine in a stoichiometry of about one vitamin E per 10 phospholipid molecules. Vitamin E partitions into domains of phosphatidylcholine in model membranes formed from mixtures of phosphatidylcholine and phosphatidylethanolamine irrespective of whether the phosphatidylcholine is in the fluid or gel phase. The creation of domains enriched in vitamin E in membranes is not consistent with an antioxidant function and effects on membrane structure and stability indicate other roles of the vitamin.     

Effect of hyperoxia on phosphatidylcholine synthesis, secretion, uptake and stability in the newborn rabbit lung

The effects of breathing greater than 95% oxygen from birth for 48 h of life on surfactant phosphatidylcholine synthesis and secretion, as well as uptake and stability of exogenous phosphatidylcholine were studied using rabbit lung tissue slices. Lung slices from animals breathing greater than 95% oxygen for 48 h exhibited a decreased rate of [14C]phosphatidylcholine release (30%) in comparison to lung slices from air-exposed controls. In vitro incorporation of [14C]choline into phosphatidylcholine was decreased by a similar amount in lung slices from pups exposed to greater than 95% oxygen. Uptake of exogenous [14C]phosphatidylcholine by lung slices from hyperoxic-exposed and control groups was not different, and the stability of extracellular phosphatidylcholine was likewise unaffected by hyperoxia. Turnover of labelled phosphatidylcholine taken up by tissue slices from medium was apparently decreased in association with hyperoxic exposure. These results are consistent with multiple sites of effect of hyperoxia on the pulmonary surfactant system in the newborn. These effects of hyperoxia on the lung surfactant system occur at a time of critical adaption to extrauterine life, and thus may have major consequences on lung function and ultimate survival of the premature infant with respiratory distress syndrome.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Cell membranes and apoptosis: role of cardiolipin, phosphatidylcholine, and anticancer lipid analogues.

The apoptotic program utilizes cellular membranes to transduce and generate operative signals. Lipids are major components of cellular membranes and have the potential to control the effectiveness of the signal by directing it to the proper location, being a source of new signals or as mediators in the response. These possible lipid functions are illustrated in the present review, focussing on the role that two different phospholipids, cardiolipin and phosphatidyl choline, play in apoptosis. Mitochondria have a central role in apoptosis, and many important aspects of the process mediated by this organelle converge through its distinctive lipid cardiolipin. Specifically, changes in cardiolipin metabolism have been detected in early steps of the death program and it is postulated (i) to mediate recruitment of pro apoptotic proteins like Bid to the mitochondria surface and (ii) to actively participate in the release of proteins relevant for the execution phase of apoptosis, like cytochrome c. Unlike the organelle specific distribution of cardiolipin, phosphatidylcholine is widely distributed among all organelles of the cell. The importance of phosphatidylcholine in apoptosis has been approached mainly through the study of the mode of action of (i) phosphatidylcholine anticancer analogues such as edelfosine and (ii) molecules that alter phosphatidylcholine metabolism, such as farnesol. The contribution of phosphatidylcholine metabolism to the apoptotic program is discussed, analyzing the experimental evidence available and pointing out some controversies in the proposed mechanisms of action.     

Multiple phase equilibria in binary mixtures of phospholipids

Approximate phase diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data presented here, as well as other earlier spin-label, calorimetric, and X-ray data. These phase diagrams represent an improvement over previous studies in that both solid phases (P_β' and L_β') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two P_β' phases that do not form a continuous range of solid solutions.     

Cell line A549 as a model of the type II pneumocyte. Phospholipid biosynthesis from native and organometallic precursors.

1. A549 is a continuous cell line derived from a human pulmonary adenocarcinoma. To evaluate the suitability of this cell line as a model of the type II pneumocyte, the morphology and the composition and biosynthesis of phosphatidylcholine was examined under control culture conditions and during fatty acid supplementation with palmitate. A number of the ultrastructural characteristics of A549 cells were similar to the in situ type II pneumocyte and were unchanged by fatty acid supplementation. The phospholipid composition of the cell line was similar to that of primary isolates of type II cells in total phosphatidylcholine, disaturated phosphatidylcholine, and palmitate and saturated fatty acid. Phospholipid biosynthetic results were also consistent with those reported for isolated type II cell models. These included: (i) the pattern of incorporation of choline, palmitate and acetate into phosphatidylcholines; (ii) the effect of palmitate supplementation, which resulted in stimulation of the rate of phosphatidylcholine biosynthesis and in increased percentage of labeled precursor in disaturated phosphatidylcholine; and (iii) the preferential synthesis from labeled choline and palmitate of a highly disaturated phosphatidylcholine in short-term incubations. 2. The incorporation of an organometallic palmitate analog, 12,12-dimethyl-12-stannahexadecanoate, into A549 cell lipids was examined and compared to that of palmitate. These date demonstrate for the first time the incorporation of an organometallic substrate into the phospholipids of a mammalian cell line. This analog substitutes selectively for the native fatty acid at a rate similar to that of the native fatty acid with no cytotoxic effects. The organotin probe, coupled with spectroscopic detection and electron microscopy, may be useful for examining ultrastructural aspects of phospholipid synthesis, translocation and assembly.     

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.     

Regulation of phosphatidylcholine metabolism by cyclic AMP in a model alveolar type 2 cell line.

The influence of cyclic AMP on the metabolism of phosphatidylcholine, the major component of pulmonary surfactant was examined in a cell line (A549) with type 2 pneumonocyte characteristics. It was found that cyclic AMP increased both the total amount of phosphatidylcholine and disaturated phosphatidylcholine as well as the incorporation of [3H]choline into these fractions. The effect was specific for cyclic AMP since 5'-AMP, adenosine, and cyclic GMP did not alter phosphatidylcholine or disaturated phosphatidylcholine levels. Cyclic AMP had no effect on phosphatidylcholine and disaturated phosphatidylcholine metabolism in another non-type 2 human epithelial cell line (MA-160). Since the ability of various cyclic AMP analogs to increase phosphatidylcholine and disaturated phosphatidylcholine levels was correlated with their ability to activate protein kinase, it seems likely that a protein phosphorylation mechanism is involved in controlling phosphatidylcholine metabolism.     

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.     

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.     

Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.     

The peptide antibiotic clavanin A interacts strongly and specifically with lipid bilayers

In this study the interaction of the antimicrobial peptide clavanin A with phosphatidylcholine bilayers is investigated by DSC, NMR, and AFM techniques. It is shown that the peptide interacts strongly and specifically with the lipids, resulting in increased order-disorder phase transition temperatures, phase separation, altered acyl chain and headgroup packing, and a drastically changed surface morphology of the bilayer. These results are interpreted in terms of clavanin-specific interactions with lipids and are discussed in the light of the different mechanisms by which clavanin A can destroy the barrier function of biological membranes.