Membrane lipids and cytosol carbohydrates in <Emphasis Type="Italic">Aspergillus niger</Emphasis> under osmotic,oxidative, and cold impact

The composition of the membrane lipids and cytosol soluble carbohydrates under three kinds of unfavorable impacts (osmotic, oxidative, and cold) was studied. Changes in the composition of the membrane lipids, specifically, increasing content of phosphatidic acids and decreasing levels of phosphatidylcholines and phosphatidylethanolamines, were the general response to the impacts. The degree of fatty acid unsaturation increased in all dominant phospholipids under osmotic shock, only in cardiolipins and phosphatidic acids under oxidative stress, and only in phosphatidylcholines under cold shock. Increased sterol content was observed only under cold and osmotic treatments. No general pattern was revealed in the composition of cytosol carbohydrates in response to stresses. Oxidative stress had almost no effect on the carbohydrate composition, while osmotic and cold treatments resulted in increased glycerol content and decreased total carbohydrate content. The mechanisms of fungal response to various stress impacts are discussed.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Lipid metabolism in Aspergillus niger under conditions of heat shock

The processes of lipid synthesis and decomposition in Aspergillus niger under conditions of heat shock (HS) were studied in a pulse-chase experiment with ¹⁴C-labeled sodium acetate. HS (60 min) resulted in the synthesis of phospholipids and sphingolipids intensified compared to the control, as was evident from incorporation of the labeled substrate. The same pattern was observed for neutral lipids, especially for triacylglycerides, while incorporation of the label into sterols remained almost the same. Further cultivation for 3 h in the medium without the labeled substrate resulted in a significant decrease of the label content in the membrane lipids of both the control and the experiment, although under HS conditions this decrease was much more pronounced, especially for phosphatidylcholines and phosphatidylethanolamines. A threefold increase of the label content in phosphatidic acids was observed only under HS conditions. These results indicate more intense metabolism of the membrane lipids under heat shock and suggest the degradation of the major cell phospholipids as the factor responsible for the increased level of phosphatidic acids in A. niger mycelium.     

In vivo metabolism of rat submaxillary gland phospholipids]

The metabolism of the phospholipids of the submaxillary gland are studied after intraperitoneal injections of [32P]-orthophosphate in several groups of rats. The gland contains mainly phosphatidylcholines and phosphatidylethanolamines and in smaller quantities phosphatidylinositols, phosphatidylserines, sphingomyelins, polyglycerophosphatides, lysophosphatidylcholines and phosphatidic acids. The specific activities of the phospholipids measured in relation to the time (1/2 hour, 1, 2 and 3 hours) show a strong incorporation in the phosphatidylinositols then in the phosphatidylcholines. The other phospholipids have lower activities. The specific activities of the phospholipids measured one hour after injection of different pharmacodynamic agents show the phosphatidylinositols, phosphatidylcholines, sphingomyelins and lysophosphatidylcholines are altered. Acetylcholine increases their turnover, Atropine reduces it, and the addition of atropine counteracts the effect of acetylcholine in all these phospholipids.     

Acyl chain interdigitation in saturated mixed-chain phosphatidylcholine bilayer dispersions

The molecular packing of various fully hydrated mixed-chain phosphatidylcholines was studied by X-ray diffraction and electron microscopy. All of the mixed-chain phosphatidylcholines under study were shown to adopt a lamellar or bilayer form in aqueous media. The bilayer thickness of these mixed-chain phosphatidylcholines was determined from the lamellar repeat distance in the small-anglé X-ray diffraction region by controlled swelling experiments. At T greater than Tm, the bilayer thickness of C(18):C(12)PC and C(18):C-(10)PC is found to be comparable to that of C(14):C(14)PC. In contrast, the bilayer thickness of these highly asymmetric phosphatidylcholines is considerably less than that of the symmetric C(14):C(14)PC at temperatures below Tm. Moreover, the wide-angle X-ray diffraction patterns taken at T less than Tm consist of at least two sharp reflections at 4.2 and 4.6 A. These X-ray diffraction data suggest that these highly asymmetric mixed-chain phospholipids, in excess water, form mixed interdigitated bilayers in the gel state and that the acyl chain packing in the gel-state bilayer is not hexagonal. The freeze-fracture planes of these mixed-chain phosphatidylcholines are discontinuous at T less than Tm, supporting the conclusion drawn from X-ray diffraction data that these highly asymmetric phosphatidylcholines form interdigitated bilayers at temperatures below Tm. The molecular packing of fully hydrated C(18):C(14)PCs in bilayers is distinctively different from that of C(18):C(10)PCs or C(18):C(10)PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiolipins and biomembrane function.

Evidence is discussed for roles of cardiolipins in oxidative phosphorylation mechanisms that regulate State 4 respiration by returning ejected protons across and over bacterial and mitochondrial membrane phospholipids, and that regulate State 3 respiration through the relative contributions of proteins that transport protons, electrons and/or metabolites. The barrier properties of phospholipid bilayers support and regulate the slow proton leak that is the basis for State 4 respiration. Proton permeability is in the range 10(-3)-10(-4) cm s-1 in mitochondria and in protein-free membranes formed from extracted mitochondrial phospholipids or from stable synthetic phosphatidylcholines or phosphatidylethanolamines. The roles of cardiolipins in proton conductance in model phospholipid membrane systems need to be assessed in view of new findings by Hübner et al. [313]: saturated cardiolipins form bilayers whilst natural highly unsaturated cardiolipins form nonlamellar phases. Mitochondrial cardiolipins apparently participate in bilayers formed by phosphatidylcholines and phosphatidylethanolamines. It is not yet clear if cardiolipins themselves conduct protons back across the membrane according to their degree of fatty acyl saturation, and/or modulate proton conductance by phosphatidylcholines and phosphatidylethanolamines. Mitochondrial cardiolipins, especially those with high 18:2 acyl contents, strongly bind many carrier and enzyme proteins that are involved in oxidative phosphorylation, some of which contribute to regulation of State 3 respiration. The role of cardiolipins in biomembrane protein function has been examined by measuring retained phospholipids and phospholipid binding in purified proteins, and by reconstituting delipidated proteins. The reconstitution criterion for the significance of cardiolipin-protein interactions has been catalytical activity; proton-pumping and multiprotein interactions have yet to be correlated. Some proteins, e.g., cytochrome c oxidase are catalytically active when dimyristoylphosphatidylcholine replaces retained cardiolipins. Cardiolipin-protein interactions orient membrane proteins, matrix proteins, and on the outerface receptors, enzymes, and some leader peptides for import; activate enzymes or keep them inactive unless the inner membrane is disrupted; and modulate formation of nonbilayer HII-phases. The capacity of the proton-exchanging uncoupling protein to accelerate thermogenic respiration in brown adipose tissue mitochondria of cold-adapted animals is not apparently affected by the increased cardiolipin unsaturation; this protein seems to take over the protonophoric role of cardiolipins in other mitochondria. Many in vivo influences that affect proton leakage and carrier rates selectively alter cardiolipins in amount per mitochondrial phospholipids, in fatty acyl composition and perhaps in sidedness; other mitochondrial membrane phospholipids respond less or not at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Heat shock response in the thermophilic fungus Rhizomucor miehei

Changes in the composition of the membrane lipids and cytosol carbohydrates of the thermophilic fungus Rhizomucor miehei in response to heat shock were studied. Under optimal conditions (41–43°C), high trehalose content (8–11%) was found at all stages of growth of submerged culture. Heat shock (51–53°C) for 1 h did not result in enhanced trehalose synthesis, while increase in shock duration to 3 h resulted in a significant increase in trehalose content. The share of sterols and phosphatidic acids in the membrane lipids increased, while the share of phosphatidylcholines and phosphatidylethanolamines decreased. These processes resulted in increased content of non-bilayer lipids, while the unsaturation degree of the fatty acids of the major phospholipids did not decrease. Comparison of resistance to lethal heat shock in the control and experimental variants of R. miehei revealed that this thermophilic fungus exhibited no acquired heat resistance.     

Formation of inverted hexagonal phase in SDPE as observed by solid-state (31)P NMR

Docosahexaenoic acid (DHA), the longest and most unsaturated fatty acid commonly found in biological membranes, is known to affect various membrane properties. In a variety of cell membranes, DHA is primarily incorporated in phosphatidylethanolamines, where its function remains poorly understood. In order to understand the role of DHA in influencing membrane structure, we utilize (31)P NMR spectroscopy to study the phase behavior of 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE) in comparison to 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine (POPE) from 20 to 50 degrees C. Spectra of SDPE phospholipids show the formation of inverted hexagonal phase (H(II)) from 20 to 50 degrees C; in contrast, POPE mutilamellar dispersions exist in a lamellar liquid-crystalline phase (L(alpha)) at the same temperatures. The ability of SDPE to adopt nonbilayer phases at a physiological temperature may indicate its role in imparting negative curvature stress upon the membrane and may affect local molecular organization including the formation of lipid microdomains within biological membranes.     

Phospholipase Dalpha3 is involved in the hyperosmotic response in Arabidopsis

Rapid activation of phospholipase D (PLD), which hydrolyzes membrane lipids to generate phosphatidic acid (PA), occurs under various hyperosmotic conditions, including salinity and water deficiency. The Arabidopsis thaliana PLD family has 12 members, and the function of PLD activation in hyperosmotic stress responses has remained elusive. Here, we show that knockout (KO) and overexpression (OE) of previously uncharacterized PLDalpha3 alter plant response to salinity and water deficit. PLDalpha3 uses multiple phospholipids as substrates with distinguishable preferences, and alterations of PLDalpha3 result in changes in PA level and membrane lipid composition. PLDalpha3-KO plants display increased sensitivities to salinity and water deficiency and also tend to induce abscisic acid-responsive genes more readily than wild-type plants, whereas PLDalpha3-OE plants have decreased sensitivities. In addition, PLDalpha3-KO plants flower later than wild-type plants in slightly dry conditions, whereas PLDalpha3-OE plants flower earlier. These data suggest that PLDalpha3 positively mediates plant responses to hyperosmotic stresses and that increased PLDalpha3 expression and associated lipid changes promote root growth, flowering, and stress avoidance.     

Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL

Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.     

Relationship between losses in cytochrome oxidase activity and peroxidation of monosaturated phosphatidylcholines and phosphatidylethanolamines.

Incubation of inner mitochondrial membranes from rat liver in the presence of inducers of peroxidation reactions, such as ascorbate or cysteine, produced a large loss in cytochrome oxidase activity parallel to the disappearance of phosphatidylcholine and phosphatidylethanolamine molecular species, which contained a saturated and an unsaturated fatty acid. The loss in enzyme activity was unrelated to alterations in other species of these phospholipids or other ones. These results may reflect the existence of specific associations within the membrane between cytochrome oxidase and monosaturated phosphatidylcholines and/or phosphatidylethanolamines.     

The structure of plant vacuolar membranes according to infrared spectroscopy

The structure of the vacuolar membrane (tonoplast) was studied in red beet roots by IR spectroscopy. The vacuolar membrane was shown to be composed of highly ordered lipids which form regions of free liquid lipid bilayer loosely bound to integral proteins. The prevalence of polar lipids in the tonoplast is responsible for the high elasticity and fluidity of the membrane. The presence of alpha-tocopherol in the tonoplast membrane accounts for a high antioxidant activity of the membrane. Integral proteins are immersed into the liquid matrix of the lipid bilayer to a different extent. Examination of the temperature effect on the kinetics of the hydrogen-deuterium exchange in integral membrane proteins showed that the efficient energy of the hydrogen exchange activation was 24 +/- 4 kcal/mol at 19-40 degrees C and increased to 54 kcal/mol at 40-50 degrees C because of the thermal denaturation of proteins. The secondary structure of integral membrane proteins is characterized by a high content of alpha-helices (53%) which decreased to 8% after the extraction of lipids.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Thermal phase transitions in deuterated lecithin bilayers.

Differential Thermal Analyses and Optical Density measurements show that perdeuteration of the fatty acid residues in phosphatidylcholines causes a 4–5°C decrease in the phase transition temperatures of bilayer dispersions prepared from these deuterated phospholipids. The implications of these findings on the use of deuterated phospholipids in membrane research will be briefly discussed.     

Phase stability of phosphatidylcholines in dimethylsulfoxide solutions.

The temperature dependence of the phase stability of dispersions of dimyristoyl, dipalmitoyl, and distearoyl derivatives of phosphatidylcholines in excess aqueous dimethylsulfoxide has been examined by differential scanning calorimetry and synchrotron x-ray diffraction methods. There was a close correlation between the enthalpic transitions and the structural changes associated with the pre- and main transitions of the phospholipids in the range of concentrations up to mole fractions of dimethylsulfoxide in water of 0.1333. The temperature of the pre- and main transitions of the three phospholipids were found to increase linearly with increasing mole fraction of dimethylsulfoxide. The difference in phase stability between the lamellar gel and ripple phases induced by increasing dimethylsulfoxide concentration resulted in disappearance of the ripple phase and direct transition between lamellar gel and lamellar liquid-crystal phases. The effect of changing the properties of the solvent by the addition of dimethylsulfoxide on the dimensions of dipalmitoylphosphatidylcholine and solvent layers of the bilayer repeat structure has been determined from electron density distribution calculations. The lamellar repeat spacing recorded at 25 degrees C decreased from 6.36 nm in aqueous dispersion to 6.04 nm in a dispersion containing a mole fraction of 0.1105 dimethylsulfoxide. The results indicate that dipole interactions between solvent and phospholipid and dielectric properties of the solvent are important factors in the determination of the structure of saturated phosphatidylcholines.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

Variation in corn (Zea mays L.) for fatty acid compositions of triglycerides and phospholipids

The percentage of linoleic acid in corn germ oil of three crosses, C103D × B73, C103D×B84, and T220×H51, and their reciprocals was investigated. Corn germ oil from F₂, F₃, and backcrossed generations was also examined. More than one gene locus appeared to be involved in conditioning the linoleic acid content in these crosses. Strong maternal effects were exhibited in the F₁'s. Genotype also superimposed variations in fatty acid compositions within the characteristic lipid class patterns of the phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Fatty acid placements in triglycerides, digalactosyldiglycerides, and phospholipids of one inbred, H51, were determined by lipase and phospholipase hydrolysis. The overall pattern of placement showed that the fatty acids at the 1 position were predominately saturated and those at the 2 position were predominately unsaturated, but the fatty acid distribution was different for each individual lipid class. The molecular species of the phosphatidylcholines and phosphatidylethanolamines were separated by silver nitrate thin-layer chromatography. The major differences in the molecular species were a higher level of the dienoic-dienoic species and a lower level of the monoenoic-monoenoic species in the phosphatidylethanolamines than in the phosphatidylcholines.     

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.     

Characteristics of fatty acid composition of lipids in higher plant vacuolar membranes

The fatty acid composition of vacuolar membrane lipids from plant storage tissues and their genesis have been studied. A high content of unsaturated fatty acids (up to 77%) was observed in lipids of these membranes. Linoleic acid prevailed in vacuolar lipids of carrot and red beet (54.2 and 44.2%, respectively). Linolenic acid prevailed in vacuolar lipids of garden radish and turnip (39.7 and 33.9%, respectively). Regarding saturated fatty acids, vacuolar lipids of garden radish, carrot, and red beet contained predominantly palmitic acid (up to 20-24%). Unsaturated fatty acids, petroselinic (C18: 1omega12), cis-vaccenic (C18: 1omega7), hexatrien-7,-10,-13-oic (C16:3omega3) and others, were observed in vacuolar lipids of roots. These acids are usually synthesized in chloroplasts, and their presence in vacuolar lipids can be associated either with the transport of metabolites to the vacuole, or with endocytosis during vacuolar formation in the plant cell. The specific features of fatty acid composition of tonoplast lipids apparently are closely related to the tonoplast unique fluidity and mobility required for running osmotic processes in the cell and for forming transport protein assemblies.