Separation of brain phosphatidylserines according to degree of unsaturation by thin-layer chromatography

Phosphatidylserines (PS) have been prepared from bovine brain using DEAE column chromatography. A method involving AgNO₃-impragnated silica gel H thin-layer chromatography is presented for separating intact PS according to the degree of unsaturation of their fatty acids. A detailed analysis was made of the fatty acid composition of the various fractions using gas chromatography. Some data are presented on the composition of molecular species of PS in bovine brain. The two main molecular species found in cerebral cortex are tentatively assigned the structures of 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine.     

Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the Black Sea fish

The phospholipid and the fatty acid composition of the main phospholipids families of erythrocyte plasma membranes was studied in two species of cartilaginous fish: the common thrasher (Raja clavata L.) and the common stingray (Dasyatis pastinaca) and three bony fish species: the scorpion fish (Scorpaena porcus L.), the smarida (Spicara flexuosa Raf.), and the horse mackerel (Trachurus mediterraneus ponticus Aleev). It was shown that in the studied fish, 70.0-80.0 % of all membrane phospholipids were composed of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine, monophosphoinositide, and sphingomyelin were minor components whose content in the erythrocyte membrane fluctuated from 3.0 % to 13.0 %. The fatty acid phospholipids composition was represented by a large specter of acids. From saturated acids, basic for plasma membranes are palmitic (C16: 0) and stearic (C18: 0) acids. From unsaturated acids, the larger part belong to mono-, tetra-, penta-, and hexaenoic acids in fish phospholipids. The calculation of the double bond index and of the unsaturation coefficient showed difference in the deformation ability of erythrocyte membranes of the studied fish.     

Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the black sea fish

The phospholipid and the fatty acid composition of the main phospholipid families of erythrocyte plasma membranes was studied in two species of cartilaginous fish: the common thrasher (Raja clavata L.) and the common stingray (Dasyatis pastinaca) and in three bony fish species: the scorpion fish (Scorpaena porcus L.), the smarida (Spicara flexuosa Raf.), and the horse mackerel (Trachurus mediterraneus ponticus Aleev). It was shown that in the studied fish, 70.0–80.0% of all membrane phospholipids were composed of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine, monophosphoinositide, and sphingomyelin were minor components whose content in the erythrocyte membrane fluctuated from 3.0% to 13.0%. The fatty acid phospholipids composition was represented by a large specter of acids. From saturated acids, basic for plasma membranes are palmitic (C16: 0) and stearic (C18: 0) acids. From unsaturated acids, the larger part belongs to mono-, tetra-, penta-, and hexaenoic acids in fish phospholipids. The calculation of the double bond index and of the unsaturation coefficient showed difference in the deformation ability of erythrocyte membranes of the studied fish.     

Application of proton NMR spectroscopy in the study of lipid metabolites in a rat hepatocarcinogenesis model

Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.     

The role of defensin fragment in the regulation of fatty acid composition of membrane phospholipids in epithelial-like cells

It is shown that a tetrapeptide fragment of defensin does not alter the phospholipid composition in the membranes of CHO-K1 cells but regulates the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine (PEA), phosphatidylserine (PS), and phosphatidylinositol (PI). Incubation of the cells in the presence of this tetrapeptide resulted in modification of unsaturated fatty acid composition in the studied phospholipids. The content of monoenoic (mainly C18 : 1ω9) and/or dienoic (C18 : 2ω6) fatty acids increased, while the level of polyenoic fatty acids decreased. It was found that in the polyenoic fatty acid group of the PEA, PS and PI molecules, the ω3-/ω6-acid ratio decreased mainly due to the lower content of long-chain ω3-acids with 20 and/or 22 carbonic atoms. The possible role of this peptide in inhibition of the activity of Δ6- and Δ5-desaturases involved in the synthesis of long-chain polyenoic fatty acids, the quantitative alteration of which in phospholipids influences physicochemical parameters in cell membranes, is discussed.     

A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina

Dipolyunsaturated phosphatidylcholines from bovine retina contain a whole series of unusual fatty acids. Methyl esters from these acids are very strongly retained on polar and nonpolar gas-liquid chromatography stationary phases. On thin layers of silica-AgNO3, they separate as tetra-, penta-, and hexaenoic fatty acid methyl esters. After hydrogenation, the three polyunsaturated fractions give the same series of saturated methyl esters, having 20 (or 22)-36 carbon atoms. High pressure liquid chromatography, as well as gas-liquid chromatography, indicates that the new components of the three fractions are even-carbon homologs of well known polyenoic fatty acids of the n-6 and n-3 families, since they behave as series of 20-36-carbon tetraenoic (n-6), pentaenoic (n-3 and n-6), and hexaenoic (n-3) fatty acids. Their occurrence in phospholipid molecules also having docosahexaenoate (22:6) explains the separation of major dipolyunsaturated phosphatidylcholines from retina into dodecaenoic, undecaenoic, and decaenoic fractions after argentation thin layer chromatography. Using high pressure liquid chromatography, the latter are resolved into individual species having 10-12 double bonds and 42-58 carbon atoms. The unusual PCs are thus endowed not only with the highest degree of unsaturation, but with the longest hydrocarbon chains yet reported for vertebrate glycerophospholipids. It is shown that phosphatidylcholines containing the novel fatty acids are highly concentrated in photoreceptor membranes and that they occur in the retina of vertebrates so distant in evolution as fish, birds, and various mammals.     

Fatty acid composition and unsaturated of intracellular membrane phospholipids from rat liver and hepatoma 27]

The fatty acid composition of phospholipids of mitochondria and microsomes from rat liver and hepatoma 27 was investigated. Basing on the fatty acid and phospholipid composition the unsaturation of the lipid bilayer of the intracellular membranes was calculated. The unsaturation of the phospholipids of the hepatoma mitochondria and microsomes was found to be much lower than that of the corresponding rat liver membranes. The lipid bilayer of the rat liver and hepatoma plasma membranes was shown to be more saturated than that of the intracellular membranes.     

Glucosyltransferase activity in mitochondria. Biosynthesis of glucosyl-phosphoryl-dolichol in inner mitochondrial membranes

1. Inner mitochondrial membranes are able to transfer [14C]glucose from UDP-[14C]glucose onto dolichylmonophosphate. 2. Synthesis of dolichyl-phosphoryl-glucose takes place only in the presence of exogenous dolichyl-monophosphate loaded into phospholipid vesicles. 3. Neutral phospholipids interact preferentially with the membrane-bound enzyme. The effect of phospholipids is not related to the length of fatty acid chains but a correlation between the activation and the degree of unsaturation of fatty acid chains has been found. 4. This enzyme required divalent cations for activity. Such a requirement might be related to lipid-protein interactions which favour a suitable conformation of glycosyltransferase.     

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.     

Proton translocation by ATPase and bacteriorhodopsin.

Stable membrane proteins and lipids are convenient to study biomembranes. Two stable proton translocating proteins were purified and reconstituted into vesicles capable of proton translocation. One was a thermostable ATPase (TF0-F1) of thermophilic bacterium PS3 and the other was rhodopsin of Halobacterium halobium. TF0-F1 was composed of a proton pump moiety (TF1) and a proton channel moiety (TF0). TF1 was the first membrane ATPase which was crystallized and reconstituted from its five polypeptides. Like TF0 and TF1, the rhodopsin in purple membrane was highly stable against dissociating agents, acids and alkali. Phospholipids of these biomembranes were also stable and contained no unsaturated fatty acyl groups. The molecular species of the phospholipids of PS3 were determined by mass chromatography. Measurements were made of the difference in electrochemical potential of protons (deltamicronH+) across the membrane of the reconstituted vesicles. The deltamicronH+ attained was 312 mV in TF0-F1 vesciles and was 230 mV in the rhodopsin vesicles. To conclude that electron transport components are not necessary for ATP synthesis in energy yielding biomembranes, two experiments were performed: The ATP synthesis was observed i) on acid-base treatment of TF0-F1 vesicles, and ii) on illumination of the rhodopsin-TF0-F1 vesicles.     

Changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of Neurospora crassa

Growth of a choline requiring auxotroph of Neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. Whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. In these lipids, choline deprivation produced little change in fatty acid profiles of phosphatidylethanolamine, whereas changes in fatty acids of phosphatidylcholine and acidic phospholipids resulted in increased levels of unsaturation at both growth temperatures. Microsomal phospholipids also showed fatty acid variability with sharp decreases in phosphatidylcholine unsaturates and increases in acidic phospholipid unsaturated fatty acids at low growth temperatures. Fluorescence polarization of 1,6-diphenylhexatriene in vesicles formed from total cellular and microsomal lipids showed that choline deprivation produces changes in thermotropic properties in the lipids in deprived cultures at either growth temperature. The effective differences in fluorescence polarization between choline-deprived and supplemented cultures grown at a given temperature were found to be comparable to those produced by temperature acclimation in normally growing cultures over a temperature range of 22 K.     

Somaclonal variation and chilling tolerance improvement in rice: Changes in fatty acid composition

The relationship between chilling tolerance of six rice cultivars – Facagro 57, Facagro 76, Fujisaka 5, Kirundo 3, Kirundo 9 and IR64 -and the fatty acid composition in total lipids, phospholipids, galactolipids and neutral lipids from leaves was studied. Higher double bond index and proportions of linolenic acid in the phospholipid and galactolipid classes were related to cultivar chilling tolerance, but this was not so for the total lipids nor the neutral lipid class. The somaclonal families derived from Facagro 76, Kirundo 3 and Kirundo 9 that showed enhanced chilling tolerance as compared to their original parental cultivar were analyzed for fatty acid composition in phospholipids and galactolipids from leaves. Altered proportions in fatty acid composition in phospholipids, galactolipids or both were found in the somaclonal families derived from Facagro 76 and Kirundo 9, but not from Kirundo 3. These changes most usually resulted in higher double bond index and higher proportions in linoleic and linolenic acids which were related either to lower ratio of C16 to C18 fatty acids or to higher unsaturation in the C18 fatty acid fraction. Different mechanisms thus seem to be implicated in the altered fatty acid composition of somaclones, which may be related to the chilling tolerance improvement of some somaclonal families.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

The role of the collagen tripeptide fragment GER in the adhesion activation and modification of fatty acid composition in membrane phospholipids of CHO-K1 cells

It has been found that multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications in fatty acid composition of cell membrane phospholipids. Cell incubation with the synthetic peptide increases the unsaturation indexes of phosphatidylcholin (PC), phosphatidylethanolamine (PEA) and phosphatidylinositol (PI). Arachidonic (C20:4omega6) acid is mainly contributed to the increased unsaturation index of PI. In the case of PC and PEA not only arachidonic acid but also other unsaturated fatty acids: docosatetraenoic (C22:4omega6), docosapentaenoic (C22:5omega3) and docosahexaenoic (C22:6omega3) acids are implicated in the index increasing. Besides, the elevation of relative content of molecules with polyenoic fatty acids in the group of PI molecules is accompanied by decrease in monoenoic fatty acids caused mainly by decrease in the oleic (C18:1) acid level. The role of the investigated peptide: 1) in the activation of cell adhesion as a regulator of active or non active state of integrin receptors: 2) in the alterations of fatty acid composition in main classes of phospholipids as modulator of fluidity level in annular lipid zones around these adhesive molecules is discussed.     

Fatty acid composition of phospholipids of myelin and synaptosomal proteolipid complexes from vertebrate brain.

1. Fatty acid composition of five main phospholipids of vertebrate brain myelin and synaptosomal proteolipids and membranes was studied. 2. Higher content of monoenoic and lower content of saturated and polyenoic fatty acids was found to be characteristic of phospholipids from myelin and myelin proteolipids as compared to phospholipids from synaptosomal proteolipids and membranes of vertebrates (from fishes to mammalians). Fatty acid composition of phospholipids of proteolipid complexes and of the membranes, from which they were isolated, were found to be similar in various species studied. 3. Microviscosity was found to be higher in myelin as compared to synaptosomal membranes of frog Rana temporaria and in rabbit Lepus cuniculus. It appears to be due to the difference in proteolipid content and in lipid composition of myelin and synaptosomal membranes.     

Interplay of unsaturated phospholipids and cholesterol in membranes: effect of the double-bond position

The structural and dynamical properties of lipid membranes rich in phospholipids and cholesterol are known to be strongly affected by the unsaturation of lipid acyl chains. We show that not only unsaturation but also the position of a double bond has a pronounced effect on membrane properties. We consider how cholesterol interacts with phosphatidylcholines comprising two 18-carbon long monounsaturated acyl chains, where the position of the double bond is varied systematically along the acyl chains. Atomistic molecular dynamics simulations indicate that when the double bond is not in contact with the cholesterol ring, and especially with the C18 group on its rough β-side, the membrane properties are closest to those of the saturated bilayer. However, any interaction between the double bond and the ring promotes membrane disorder and fluidity. Maximal disorder is found when the double bond is located in the middle of a lipid acyl chain, the case most commonly found in monounsaturated acyl chains of phospholipids. The results suggest a cholesterol-mediated lipid selection mechanism in eukaryotic cell membranes. With saturated lipids, cholesterol promotes the formation of highly ordered raft-like membrane domains, whereas domains rich in unsaturated lipids with a double bond in the middle remain highly fluid despite the presence of cholesterol.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.     

Proton translocating ATPase of a thermophilic bacterium. Morphology, subunits, and chemical composition.

1. A stable membrane-bound ATPase [EC 3.6.1.3] (TF0-F1) capable of proton translocation in reconstituted vesicles was purified from the thermophilic bacterium PS3 cultured in medium containing L-[U-14C]amino acids. 2. TF0-F1 was composed of a catalytic moiety (TF1) and a hydrophobic moiety (TF0). TF1 contained 3 polypeptide chains with molecular weights of 56,000, 3 of 53,000, 1 of 32,000, 1 of 15,500, and 1 of 11,000. TF0 contained 1 chain of 19,000, 2 of 13,500, and 5 of 5,400 daltons. TF1 was dissociated into subunits much less readily than F1. 3. TF1 consisted of 95A particles arrayed in hexagonal microcrystals. TF0-F1 consisted of a sphere (TF1) and a stalk plus base (TF0) which was buried in the membrane of the proton translocating vesicles. 4. Vesicles capable of energy transformation were formed when TF1 came in contact with the surface of liposomes containing TF0. On addition of phospholipids, the helix content of TF0 increased 3-fold. The role of F0 in forming channels for protons is discussed. 5. The amino acid compositions of TF0, TF1, and TF0-F1 were compared. TF0 was not hydrophobic, despite its interaction with phospholipids. The phospholipid composition and other properties of the proton translocating vesicles were examined. Vesicles reconstituted from a mixture of phosphatidylethanolamine, phosphatidylgly-cerol, and cardiolipin in the same ratio as in the membranes had the highest activity.     

Toxicity of activated oxygen: lack of dependence on membrane unsaturated fatty acid composition

Membrane unsaturated fatty acid oxidation has been suggested as a mechanism of toxicity for a variety of activated oxygen species. We have tested this hypothesis by manipulating the fatty acid composition of an Escherichia coli mutant that is unable to synthesize unsaturated fatty acids. To provide a wide range of susceptibility to membrane oxidation we have replaced the naturally occurring monoenoic acyl chains with cyclopropanes to greatly reduce the unsaturation level and with linoleate to increase the membrane unsaturation. These cultures were treated with ozone, hydrogen peroxide, singlet oxygen and paraquat. In no case was there substantial protection from toxicity afforded by cyclopropanes nor was there enhancement of toxicity to cells with the polyunsaturated membranes. We suggest, therefore, that oxidation of membrane unsaturated fatty acids is not an essential component of the toxicity to E. coli of active oxygen species.