Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.     

Independent synthesis of phospholipid and the intrinsic proteins of the sarcoplasmic reticulum

The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcoplasmic reticulum was investigated in differentiating L6 cells in culture. The rates of lipid synthesis and turnover in L6 showed no large variations over the course of differentiation from myoblasts to myotubes while the rate of synthesis of the sarcoplasmic reticulum Ca2+-ATPase steadily increased. Removal of choline from the culture medium after the onset of fusion resulted in a 2-fold inhibition of phosphatidylcholine (PC) synthesis and a 40-50% reduction in total cellular PC content within 36 h. The synthesis and content of phosphatidylethanolamine also declined subsequent to the effect on PC. The amount of newly synthesized phospholipid in the microsomal fraction also decreased 50% in choline-deprived cells. Choline deprivation of myotubes for up to 4 days had no effect on the rates of synthesis of the Ca2+-ATPase or two intrinsic glycoproteins of 53,000 and 160,000 daltons. The newly synthesized proteins were incorporated into PC-deficient microsomal membranes. The synthesis of total cellular protein and total membrane protein was not altered, thus phospholipid:protein ratios declined 2-fold. These observations suggest that the assembly of the sarcoplasmic reticulum is not tightly coordinated with the rate of phospholipid synthesis.     

PLASMA AND PHAGOSOME MEMBRANES OF ACANTHAMOEBA CASTELLANII

Plasma membranes were isolated from the ameba Acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. The isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). The plasma membranes had very low concentrations of DNA, RNA, lipid inositol, and glycerides. Glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the whole cell. The plasma membranes were also judged to be of high purity by the absence, or very low level, of enzymatic activities considered to be indicative of other cell membranes, and by electron microscope examination. Alkaline phosphatase and 5'-nucleotidase activities were enriched in the plasma membranes 13-fold relative to the whole homogenate and had higher specific activities in the plasma membranes than in any other cell fractions. A Mg⁺⁺ adenosine triphosphatase (ATPase) was enriched sixfold in the plasma membranes relative to the whole homogenate. The phospholipids of the plasma membranes contained more phosphatidylethanolamine and phosphatidylserine and less phosphatidylcholine than did the phospholipids of the whole cells. There were differences in the fatty acid compositions of corresponding phospholipids in the plasma membranes and whole cells but no difference in the ratios of total saturated to unsaturated fatty acids. The membranes of phagosomes isolated from amebae that had ingested polystyrene latex had essentially the same phospholipid, sterol, and enzymatic composition as plasma membranes.     

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Effect of neuraminidase treatment on the topological distribution of phospholipids in chick brain microsomes

The desialylation of chick brain microsomal membranes affects the transbilayer distribution of phospholipids. When intact microsomes were treated with neuraminidase, less phosphatidylcholine and sphingomyelin could be hydrolysed with phospholipase C under experimental conditions which allowed the hydrolysis of the phospholipids of the external leaflet only. In contrast, the accessibility of phosphatidylethanolamine and phosphatidylserine to the external probes (trinitrobenzene sulfonic acid or phospholipase C) was not affected. After neuraminidase treatment of a microsomal fraction, less phosphatidylcholine, newly synthesized through the cytidine pathway, could be hydrolysed by phospholipase C, whereas the reaction of newly synthesized phosphatidylethanolamine molecules with trinitrobenzene sulfonic acid was not affected. The results suggest that in biological membranes some choline phospholipid molecules may interact with the sialyl residue of sialocompounds. This interaction may contribute to the maintenance of phospholipid asymmetry in brain membranes.     

Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance

Plasma membranes were isolated from oat (Avena sativa) roots by the phase-partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.     

Thromboplastin (tissue factor) in plasma membranes of human monocytes.

The synthesis of thromboplastin, a potent trigger of blood coagulation, can be induced in human peripheral blood monocytes. Indirect evidence suggests that newly synthesized thromboplastin becomes in part available on the cell surface. We have attempted to study the localization and availability of thromboplastin more directly by isolating plasma membranes from isolated human peripheral blood monocytes. The specific activities of the plasma membrane markers increased 16-22-fold in these preparations with a recovery of about 15%. The contamination by mitochondria, lysosomes, nuclei and endoplasmic reticulum was low as estimated by marker enzymes and electron microscopy. In both unstimulated and stimulated monocytes thromboplastin was largely recovered in this plasma membrane fraction, providing direct evidence for its membrane localization. Phospholipase C (E.C. 3.1.4.3) is a potent inactivator of thromboplastin through its hydrolysis of the phospholipids necessary for thromboplastin activity [Otnaess, Prydz, Bjørklid & Berre (1972) Eur. J. Biochem. 27, 238-243]. About 70% of the total membrane thromboplastin activity was inactivated when whole cells were treated with phospholipase C and the membranes subsequently isolated. Following stimulation to induce thromboplastin synthesis, the plasma membranes showed a shift in their relative content of phosphatidylcholine and phosphatidylethanolamine consistent with a transmethylation process.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : II. The Origin and Fate of Glycerol3H-Labeled Phospholipids of Cellular Membranes

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-³H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-³H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.     

Phospholipid composition of substrate adhesion sites of normal, virus-transformed, and revertant murine cells.

The phospholipid composition of cell-substratum adhesion sites, obtained after EGTA-mediated detachment of cells from the tissue-culture substratum, was determined for [32P]orthophosphate radiolabeled Balb/c 3T3, SV40-transformed (SVT2), and concanavalin A selected revertant variant cell lines. All of the major phospholipid classes were found in the substrate-attached material, but there was an enrichment for specific phospholipid species in this adhesive material as compared to whole-cell and surface-enriched membranes. The phospholipid composition was remarkable similar for the whole-cell and surface-enriched membrane fractions from the three cell lines. However, pronounced differences in the phospholipid composition of the adhesion sites were observed as a result of viral transformation--SVT2 sites were clearly enriched in phosphatidylethanolamine and depleted in phosphatidylcholine when compared to 3T3 sites. This alteration in adhesion site phospholipids of transformed cells reverted to 3T3-like values in the adhesive material of revertant cells. The composition of adhesive material of newly attaching cells was also examined to differentiate compositional differences between "footpad" adhesion sites and "footprints", adhesive material pinched off from the posterior of cells as they move across the substratum. Pulse and pulse-chase analyses of the [32P]phospholipids revealed some differences in synthesis and turnover rates in the three cell lines; in addition, altered rates of deposition of newly synthesized material into adhesion sites of transformed cells were observed. These data afford further evidence that the cell-substratum adhesion sites are highly specialized areas of the cell surface enriched in components which are intricately involved in the adhesive process. The transformation-dependent changes in adhesion site phospholipids may help to determine the basis for the altered adhesive properties of transformed cells.     

Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.     

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

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.     

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     

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.     

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.