Positional distribution of fatty acids in lipids of the marine diatom Phaeodactylum tricornutum

The composition of fatty acids and lipids in the marine diatom, Phaeodactylum tricornutum was determined. The Lipids consisted of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphtidylinositol, triacylglycerol and minor unidentified ones. At the early stationary phase of growth, the total fatty acids were mainly 20:5, 16:1, 16:0 and 16:3. 20:5 was distributed in polar lipids, particularly in monogalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol. This fatty acid was exclusively located at the sn-1 position of the glycerol moiety in all polar lipids except for phosphatidylcholine. In phosphatidylcholine 20:5 was distributed at both the sn-1 and sn-2 positions. 16:3 was concentrated at the sn2 position of monogalactosyldiacylglycerol and trans-16:1 (n-13) was dominant at the sn-2 position of phosphatidylglycerol. C₁₈ fatty acids, the minor fatty acids in P. tricornutum, were confined to the sn-2 position of phosphatidylcholine.     

The effect of low oxygen concentration on the acyl-lipid and fatty-acid composition of the C4 plant Amaranthus paniculatus L.

Acyl lipids and their constituent fatty acids were studied in leaves, chloroplasts and bundle-sheath strands of the C₄ plant Amaranthus paniculatus L. grown under normal and 4%-oxygen-containing atmospheres. In all fractions the major lipids were found to be monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovo-syldiacylglycerol and phosphatidylglycerol. Significant quantities of phosphatidylcholine and phosphatidylethanolamine were restricted to leaves and bundle-sheath strands. All lipids, except phosphatidylglycerol where 3-trans-hexadecenoic acid was also present, contained palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. On a chlorophyll basis and compared with whole leaves, the amounts of phosphatidylcholine and phosphatidylethanolamine in bundle-sheath strands were considerably reduced. Three weeks after the change from a normal to a 4% atmospheric O₂ level, the galactolipid content, particularly in the bundlesheath strands, was enhanced. There were no significant differences in the degrees of saturationunsaturation of total acyl lipid for the plants grown in the low oxygen and normal atmospheres, although under 4% O₂ the phosphatidylglycerol contained an increased proportion of 3-trans-hexadecenoic acid at the expense of palmitic acid.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - SQDG sulphquinovosyldiacylglycerol     

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.     

Lipid metabolism in green leaves of developing monocotyledons

Lipid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivum L.) leaves. The endogenous levels of acyl lipids and their constituent fatty acids from the same leaf sections were also analysed. The principle chloroplast acyl lipids showed a relative increase in amount with the age of the leaf section. Their content of -linolenic acid also increased whereas there was little change in the amount of this acid in phosphatidylcholine and phosphatidylethanolamine, which are primarily non-chloroplastic. The content of trans-3-hexadecenoic acid in phosphatidylglycerol increased approximately 20-fold between the youngest (basal) and oldest (distal) leaf sections.The incorporation of [¹⁴C]acetate was always high into monogalactosyldiacylglycerol, phosphatidylcholine and the neutral lipid (mainly pigments) fractions. With increasing age, the neutral lipids were less well labelled. In three of the plant species but not in barley, phosphatidylglycerol was heavily labelled. Monogalactosyldiacylglycerol usually contained the highest amount of radioactivity in the middle leaf sections. Apart from these generalisations, each plant type had its own specific pattern of radiolabelling.     

Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

Combination and positional distribution of fatty acids in lipids from blue-green algae

The main glycerolipids (monogalactosyl-, digalactosyl-, sulphoquinovosyl diacylglycerol, phosphatidylglycerol) from five blue-green algae (Microcystis, Anabaena, Nostoc, Oscillatoria, Tolypothrix) were analyzed for fatty acid composition, occurrence of diglyceride species and positional distribution of fatty acids between thesn-1- andsn-2-position of glycerol. In contrast to eucaryotic plants biosynthetically closely related lipids (monogalactosyl-, digalactosyl-, trigalactosyl diacylglycerol) show nearly identical diglyceride moieties, whereas sulphoquinovosyl diacylglycerol and phosphatidylglycerol are separated from galactolipids by composition as well as occurrence of fatty acids. On the other hand the positional distribution of fatty acids in all lipids is controlled exclusively by chain length and not by degree of unsaturation with C₁₈-fatty acids at thesn-1- and C₁₆-fatty acids at thesn-2-position. These results show that in procaryotic organisms the diversity in diglyceride portions of lipids is reduced as compared to eucaryotic organisms, but nevertheless does exist.Abbreviations MGD, DGD, TGD, SQD monogalactosyl-, digalactosyl-, trigalactosyl-, sulphoquinovosyl diacylglycerol - PG phosphatidyl glycerol     

A fluorescence decay study of parinaroyl-phosphatidylinositol incorporated into artificial and natural membranes

Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membrances from Torpedo marmorata were investigated both at 4°C and 20 °C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In contrast to Torpedo membranes, the time-dependent fluorescence anisotropy of the two lipid probes in the sarcolemmal membranes showed, after an initial fast decay, a subsequent gradual increase. This phenomenon was satisfactorily analyzed by assuming two populations of probe lipids with distinct lifetimes, rotational correlation times and molecular order. The order parameter of the population with a short lifetime compared with that of phospholipid vesicles, whereas the population with a long lifetime agreed with that of the Torpedo membranes.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - PA phosphatidic acid - PE phosphatidylethanolamine - PS phosphatidylserine, PnA, cis-parinaric acid: cis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid - 2-PnA-PC 1-acyl, 2-parinaroyl-PC - 2-PnA-PI 1-acyl,2-parinaroyl-PI - DPH diphenylhexatriene - POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene - NMR nuclear magnetic resonance - ESR electron spin resonance - I parallel fluorescence intensity component - I perpendicular fluorescence intensity component - SET-buffer 0.25 M Sucrose, 1 mM EDTA, 10mM Tris-HCl, pH 7.4     

Effect of triacontanol on the lipid composition of cotton (Gossypium hirsutum L.) leaves and its interaction with indole-3-acetic acid and benzyladenine

Application of triacontanol (TRIA), a long chain aliphatic alcohol (C-30), to cotton (Gossypium hirsutum L.) leaves resulted in an increase in dry weight and an alteration in lipid composition. A significant increase in monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) was attained 24 h after TRIA treatment. However, no significant change in any of the individual phospholipids was observed. Benzyladenine (BA) treatment increased only phosphatidylcholine (PC) levels without having any effect on either glycolipids or other phospholipids. Indole-3-acetic acid (IAA) initiated no significant change in the lipid composition. Combined treatment with TRIA and BA resulted in an increase of MGDG, DGDG and PC, indicating that the individual effects of these two growth regulators were not altered.The combined treatment of IAA and TRIA did not bring about any change in the levels of MGDG and DGDG indicating that the effect of TRIA was nullified by IAA. MDGD is known to be involved in the packaging of photosystem I proteins. Whether TRIA-induced increase in dry weight which is due to the enhanced photosynthetic rate, is related to increased MGDG levels is not yet discernible.Abbreviations BA benzyladenine - DGDG digalactosyldiacylglycerol - IAA indole-3-acetic acid - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SQDG sulfoquinovosyldiacylglycerol - TRIA triacontanol     

Kinetics and localisation of haemin-induced lipoprotein oxidation

Abstract

Haemin (iron (III)-protoporphyrin IX) is a degradation product of haemoglobin in circulating erythrocytes. Haemin may play a key oxidising agent for lipoprotein oxidation in patients with haemolytic anaemia. In this study, kinetic changes in chemical composition and target sites of haemin-induced LDL and HDL oxidation were investigated. Haemin initially induced the loss of α-tocopherol, followed by accumulation of lipid hydroperoxide (LP) and alteration of core lipid fluidity. The absence of LP in HDL was explained by the antioxidant activity of PON in addition to α-tocopherol. The target site of haemin was evaluated by ESR spin labelling with 5- and 16-doxyl steric acids. In the presence of t-BuOOH and haemin, ESR signal decay of the doxyl moiety demonstrated the initiation phase and the propagation phase of lipid peroxidation. The results of the lag time and the rate of signal decay indicated that haemin is located near the 16th carbon atom of the fatty acid chain in the phospholipid layer. The analyses of motion parameters, order parameter (S) of 5-DS and rotational correlation time (τ) of 16-DS, supported the observation that the lipid properties changed near the hydrophobic region rather than at the surface region of lipoproteins. Moreover, ESR spin labelling demonstrated that haemin molecules but not iron ions caused lipoprotein oxidation. In conclusion, haemin is a potent inducer of lipoprotein oxidation, and the target site for this oxidation is near the hydrophobic core of the lipoprotein leading to the loss of antioxidant activities and changes in lipid composition and physical properties.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Effects of lipolytic enzymes on the photochemical activities of spinach chloroplasts.

1. Spinach class II chloroplasts were treated with purified potato lipolytic acyl-hydrolase and venom phospholipase A2, and their lipid degradations and the effects on the photochemical activities were followed. 2. Potato lipolytic enzyme hydrolyzed monogalactosyldiacylglycerol at a faster rate than phospholipids such as phosphatidylglycerol and phosphatidylcholine. The treatment caused a rapid decrease of Photosystem I activity, and a less change of Photosystem II activity. 3. Venom phospholipase A2 which preferentially hydrolyzed phosphatidylglycerol, caused a rapid decrease of Photosystem II activity and only a slight decrease of photosystem I activity. 4. Potato enzyme and phospholipase A2 degraded the membrane lipids of glutaraldehyde-fixed chloroplasts at a rather slightly higher rate than those of non-treated chloroplasts. 5. The results suggested a possible correlation between monogalactosyldiacylglycerol degradation and decay of Photosystem I activity and between phosphatidylglycerol degradation and decay of Photosystem II activity. A possible mechanism is discussed.     

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

THE METABOLISM OF [1-14C]ARACHIDONIC ACID IN THE NEUTRAL GLYCERIDES AND PHOSPHOGLYCERIDES OF MOUSE BRAIN1

Abstract— [1-¹⁴C]Arachidonic acid was incorporated into brain lipids with a half-life of approx. 5 min. Within 40 min after intra-cerebral injection, radioactivity was distributed mainly among the diacyl-sn-glycero-3-phosphorylcholine (45 per cent), diacyl-sn-glycero-3-phosphorylinositol (22 per cent), diacyl-sn-glycero-3-phosphorylethanolamine (14 per cent) and triacylglycerols (9 per cent). At comparable times, the proportions of radioactivity distributed in diacyl-sn-glycero-3-phosphorylserines and alkenylacyl-sn-glycero-3-phosphorylethanolamines were relatively small. Radioactivity was initially incorporated into the phosphatidio acids and diacylglycerols before labelling of the triacylglycerols and other phosphogly-cerides. The relative specific activity of diacylglycerols was maximum between 3–6 min after injection. Due to the small level of diacyl-sn-3-phosphorylinositol present in brain, its relative specific radioactivity was higher than other types of brain phosphoglycerides. Results of the experiment thus indicate that labelled arachidonic acid is an excellent precursor for metabolic studies with regard to acyl groups present in the 2-position of the phosphoglyceride molecules. Furthermore, this labelled precursor is specially useful in studies related to metabolism of diacyl-sn-glycero-3-phosphorylinositol in brain.     

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes

The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (<200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.     

Alpha-synuclein association with phosphatidylglycerol probed by lipid spin labels

Alpha-synuclein is a small presynaptic protein, which is linked to the development of Parkinson's disease. Alpha-synuclein partitions between cytosolic and vesicle-bound states, where membrane binding is accompanied by the formation of an amphipathic helix in the N-terminal section of the otherwise unstructured protein. The impact on alpha-synuclein of binding to vesicle-like liposomes has been studied extensively, but far less is known about the impact of alpha-synuclein on the membrane. The interactions of alpha-synuclein with phosphatidylglycerol membranes are studied here by using spin-labeled lipid species and electron spin resonance (ESR) spectroscopy to allow a detailed analysis of the effect on the membrane lipids. Membrane association of alpha-synuclein perturbs the ESR spectra of spin-labeled lipids in bilayers of phosphatidylglycerol but not of phosphatidylcholine. The interaction is inhibited at high ionic strength. The segmental motion is hindered at all positions of spin labeling in the phosphatidylglycerol sn-2 chain, while still preserving the chain flexibility gradient characteristic of fluid phospholipid membranes. Direct motional restriction of the lipid chains, resulting from penetration of the protein into the hydrophobic interior of the membrane, is not observed. Saturation occurs at a protein/lipid ratio corresponding to approximately 36 lipids/protein added. Alpha-synuclein exhibits a selectivity of interaction with different phospholipid spin labels when bound to phosphatidylglycerol membranes in the following order: stearic acid > cardiolipin > phosphatidylcholine > phosphatidylglycerol approximately phosphatidylethanolamine > phosphatidic acid approximately phosphatidylserine > N-acyl phosphatidylethanolamine > diglyceride. Accordingly, membrane-bound alpha-synuclein associates at the interfacial region of the bilayer where it may favor a local concentration of certain phospholipids.     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography