Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepaic cell line (WRL-68)

The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanolin vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to thein vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to thein vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.Abbreviations DPH diphenylhexatriene - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SPH sphingomyelin     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Preferential interaction of alpha-tocopherol with phosphatidylcholines in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine.

The effect of alpha-tocopherol on the structure and phase behaviour of mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine has been examined by synchrotron X-ray diffraction and freeze-fracture electron microscopy. Equimolar mixtures of fully saturated derivatives of phospholipids that show gel phase immiscibility were examined including dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoethanolamin e and distearoylglycerophosphocholine/dilauroylglycerophosphoethanolamine++ +. Analysis of the X-ray scattering intensities recorded at wide angles during heating scans of mixed aqueous dispersions containing 2.5 or 5 mol% alpha-tocopherol showed that alpha-tocopherol disordered the acyl chain packing of the phosphatidylcholine to a greater extent than the phosphatidylethanolamine component of the mixture. This suggested that alpha-tocopherol preferentially interacts with phosphatidylcholine rather than phosphatidylethanolamine, irrespective of whether this was the high or low melting point component of the mixture. The presence of 20 mol% alpha-tocopherol in either phospholipid mixture prevented gel phase separation during the prior cooling scan and no conclusions could be drawn as to the distribution of alpha-tocopherol in these mixtures.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine > cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.     

Phospholipid methyltransferase asymmetry in synaptosomal membranes

The sequential methylation of phosphatidylethanolamine to form phosphatidylcholine is carried out by two methyltransferases in rat brain synaptosomes. The first enzyme methylates phosphatidylethanolamine to form phosphatidylmonomethylethanolamine. The second enzyme methylates the monomethylated phospholipid two additional times, forming phosphatidylcholine. Experiments comparing the rate of methylation between intact and lysed synaptosomes indicate that synaptosomes accumulateS-adenosyl-l-methionine and that the first methylation takes place on the cytoplasmic side of the membrane. Studies comparing trypsin digestion of proteins in intact and lysed synaptosomes indicate that the first enzyme is localized on the cytoplasmic side of the membrane and the second enzyme faces the external surface. Phospholipase C hydrolyzed phosphatidylcholine formed by methylation, suggesting its localization in the external layer of the phospholipid bilayer. A mechanism for an enzyme-mediated flip-flop of phospholipids from the cytoplasmic side to the outer surface of the synaptosomal plasma membrane is presented.     

The distribution of alpha-tocopherol in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine

The effect of alpha-tocopherol on the structure and phase behaviour of mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine has been examined by synchrotron X-ray diffraction. Equimolar mixtures of dioleoylphosphatidylethanolamine:dioleoylphosphatidylcholine and dimyristoylphosphatidylcholine:dioleoylphosphatidylethanolamine did not show evidence of phase separation of an inverted hexagonal structure typical of alpha-tocopherol and phosphatidylethanolamine from lamellar phase. Mixed dispersions of dioleoyl derivatives of phosphatidylethanolamine:phosphatidylcholine (3:1) form a typical miscible gel phase at low temperatures but which phase separates into lamellar liquid-crystal and inverted hexagonal phases at temperatures greater than 65 degrees C. The presence of 1, 2 or 5 mol% alpha-tocopherol caused a decrease in the temperature at which the inverted hexagonal phase appears. Phase separation of non-lamellar phase from lamellar gel phase can be detected in the presence of 7.5 and 10 mol% alpha-tocopherol, indicating a limited capacity of the phosphatidylcholine to incorporate alpha-tocopherol into the lamellar domain. A partial phase diagram of the ternary mixture has been constructed from the X-ray scattering data. It was concluded that there is no preferential interaction of alpha-tocopherol with phosphatidylethanolamine in mixed aqueous dispersions containing phosphatidylcholines.     

Localizing the nitroxide group of fatty acid and voltage-sensitive spin-labels in phospholipid bilayers

The intramembrane locations of several spin labeled probes in small egg phosphatidylcholine (egg PC) vesicles were determined from the enhancement of the 13C nuclear spin lattice relaxation of the membrane phospholipid. Electron paramagnetic resonance (EPR) spectroscopy was also used to measure the relative environmental polarities of the spin labels in egg PC vesicles, ethanol and aqueous solution. The binding location of the spin label group was determined for a pair of hydrophobic ion spin labels, a pair of long chain amphiphiles, and three stearates containing doxyl groups at the 5, 10 and 16 positions. The nuclear relaxation results indicate that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid acyl chains. In addition, the spin label groups of the hydrophobic ions and long chain amphiphiles are located near the acyl chain methylene immediately adjacent to the carboxyl group. The relative polarities, determined by the EPR technique, are consistent with the nuclear relaxation results. This information, when combined with information on their electrical properties, allows for an assessment of the conformation and position of these voltage sensitive probes in membranes.     

Spin-label characterisation of the lamellar-to-hexagonal (HII) phase transition in egg phosphatidylethanolamine aqueous dispersions

The thermotropic behaviour of egg yolk phosphatidylethanolamine dispersions in excess aqueous phase has been investigated by spin label electron spin resonance spectroscopy and differential thermal analysis. Phosphatidylethanolamine isomers spin-labelled at six different positions along the acyl chain, and steroid spin labels, indicate both gel-fluid lamellar and lamellar-reverse hexagonal (HII) phase transitions, in agreement with complementary calorimetric studies. Analysis of spin label data shows that the transition to the HII phase is accompanied by an increase in conformational freedom of the acyl chain, more pronounced towards the methyl terminus, and representing an increase in the population of gauche isomers which can only be accommodated by a transition to the non-bilayer phase. Raising the bulk pH to, and above, pH 8.5 results in stabilisation of the bilayer phase and no transition to the HII phase is observed. The phosphatidylethanolamine spin labels also indicate a polarity profile which is characteristic of each phase.     

Ethanol Alters Brain Phospholipid Levels Which Correlate with Altered Brain Morphology

The effects of embryonic exposure on brain phospholipid levels were studied by injecting various concentrations of ethanol into fertile chicken eggs at 0 days of development. At 18 days of development, the levels of total phospholipids and various phospholipid classes were assayed in brain tissue and correlated to neuron densities within the cerebral hemispheres and the optic lobes. Although ethanol concentrations ranging from 0 to 3700 μm/Kg egg wt. failed to influence either total brain weight or total brain phospholipid levels, ethanol-induced changes in the levels of individual phospholipid classes were observed. When injected with 7 μm of ethanol/Kg egg wt., a 2- to 3-fold increase in brain phosphatidylethanolamine (PE) levels were observed with reduced levels of brain phosphatidylcholine (PC) and brain sphingomyelin (SP). When injected with 74 μm of ethanol/Kg egg wt., ethanol-induced increases in brain phosphatidylserine (PS) and PE were observed with ethanol-induced decreases in brain PC and SP. Cell fractionation studies demonstrated ethanol-induced increases in brain PE and PS and ethanol-induced decreases in brain PC and SP in nuclear, mitochondrial, and microsomal membranes. These ethanol-induced alterations in brain phospholipid profiles correlated with ethanol-induced reductions in neuron densities within the cerebral hemispheres and optic lobes.     

Membrane adaptation in phospholipids and cholesterol in the widely distributed,freeze-tolerant wood frog,Rana sylvatica

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to ?2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Stimulation by phospholipid vesicles of proteolysis of egg white lysozyme by chymotrypsin

Egg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid. Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.     

Alcohols inhibit adipocyte basal and insulin-stimulated glucose uptake and increase the membrane lipid fluidity

Benzyl alcohol and ethanol, at aqueous concentrations that cause local anesthesia of rat sciatic nerve, affect structural and functional properties of rat adipocytes. The data strongly suggest that structurally-intact membrane lipids are required for the proper cellular uptake of glucose and for the physiologic response of adipocytes to insulin. The structure of adipocyte membrane lipids was examined with the spin label method. Isolated adipocyte ‘ghost’ membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). Order parameters that are sensitive to the fluidity of the lipid environment of the incorporated probe were calculated from ESR spectra of labeled membranes. Benzyl alcohol and ethanol dramatically increased the fluidity of the adipocyte ghost membrane, as indicated by decreases in the polarity-corrected order parameter S. This concentration-dependent fluidization commenced at approx. 10 mM benzyl alcohol and progressively increased at all higher concentrations tested (up to 107 mM). S decreased approx. 5.7% at 40 mM benzyl alcohol, a change in S comparable in magnitude to that induced by a 6°C increase in the incubation temperature. Benzyl alcohol and ethanol inhibited basal glucose uptake in adipocytes and uptake maximally stimulated by insulin. Temperature-induced increases in membrane fluidity, detected with 1(12,3), that closely paralleled the fluidity effects of alcohols were associated only with increases in basal and insulin-stimulated glucose uptake. The contention that the membrane lipid fluidity plays a role in insulin action needs further study.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of γ-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although γ-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to γ-linolenic acid, since the γ-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of γ-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

EPR studies of phospholipid bilayers after lipoperoxidation. 1. Inner molecular order and fluidity gradient

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.     

Comparison of effects of ABA and IAA on phospholipid bilayers

The plant hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) affect the properties of phospholipid bilayers differently. IAA enhances permeability of bilayers composed of phosphatidylcholine to the non-electrolyte erythritol while ABA requires an additional phospholipid in the membrane to produce substantial enhancement. Similar conclusions are obtained by measuring hormone-induced permeability to chloride ions; IAA is effective with single component phosphatidylcholine membranes while ABA requires a second phospholipid. Erythritol permeability is shown to be pH dependent for both hormones. Although IAA is more effective at increasing erythritol permeability at pH 4 than at pH 7, both dissociated and undissociated IAA affect the process. In comparison ABA is almost totally ineffective in the dissociated form (at pH 7). Spin label electron spin resonance measurements demonstrated that neither hormone substantially disrupts acyl chain mobility within the membrane, indicating that the mechanism of permeability enhancement is not a general non-specific pertubation of membrane ordering and fluidity. Both hormones can also effect the stability of small unilamellar (sonicated) vesicles. Phosphatidylcholine vesicles are relatively stable and do not rapidly aggregate with either ABA or IAA. However, when phosphatidylethanolamine is incorporated as a minor component (10 mol%) into phosphatidylcholine vesicles ABA causes rapid aggregation while IAA has no effect. These experiments indicate that the two hormones may exhibit completely different behaviour on membranes without the requirement for specific proteinaceous receptors.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.