Showing papers on "Membrane lipids published in 1994"

Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior

Abstract: Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes. These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.

Fibril assembly and carotenoid overaccumulation in chromoplasts: a model for supramolecular lipoprotein structures.

Abstract: Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctive red color. We have shown that 95% of these pigments accumulate in chromoplasts in specific lipoprotein fibrils. In addition to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrils were reconstituted in vitro when purified fibrillin was combined with carotenoids and polar lipids in the same stoichiometric ratio found in fibrils in vivo. Antibodies directed against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-to-chromoplast transition under different conditions. A model for fibril architecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purified fibrils verified this structure. Collectively, these results suggest that the process of fibril self-assembly in chromoplasts is an example of a general phenomenon shared among cells that target excess membrane lipids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.

Molecular dynamics simulation of the gramicidin channel in a phospholipid bilayer

Abstract: A molecular dynamics simulation of the gramicidin A channel in an explicit dimyristoyl phosphatidylcholine bilayer was generated to study the details of lipid-protein interactions at the microscopic level. Solid-state NMR properties of the channel averaged over the 500-psec trajectory are in excellent agreement with available experimental data. In contrast with the assumptions of macroscopic models, the membrane/solution interface region is found to be at least 12 A thick. The tryptophan side chains, located within the interface, are found to form hydrogen bonds with the ester carbonyl groups of the lipids and with water, suggesting their important contribution to the stability of membrane proteins. Individual lipid-protein interactions are seen to vary from near 0 to -50 kcal/mol. The most strongly interacting conformations are short-lived and have a nearly equal contribution from both van der Waals and electrostatic energies. This approach for performing molecular dynamics simulations of membrane proteins in explicit phospholipid bilayers should help in studying the structure, dynamics, and energetics of lipid-protein interactions.

The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance.

Abstract: In a previous study of mutants in fatty-acid desaturation of Synechocystis PCC6803, it was demonstrated that the photoinhibition of photosynthesis at low temperature in vivo is tolerated by cells as a result of the unsaturation of glycerolipids of thylakoid membranes. Since the extent of photoinhibition of photosynthesis in vivo depends on a balance between the photoinduced inactivation and the recovery from the photoinhibited state, an examination was made of the effects of the unsaturation of membrane lipids on these processes. It appears that the unsaturation of the membrane lipids does not affect the inactivation process but accelerates the recovery process and, moreover, that the apparent increase in the photoinhibition in vivo of photosynthesis at low temperature is caused by a depressed rate of recovery at low temperature.

Stress tolerance and membrane lipid unsaturation in Saccharomyces cerevisiae grown aerobically or anaerobically.

Abstract: Saccharomyces cerevisiae cells grown either aerobically or anaerobically were tested for tolerance to a brief heat stress (52°C, 5 min) or oxidative stress (20 mM H2O2, 15 min). Tolerance was related to growth phase, in that stationary phase cells were intrinsically more resistant to heat or oxidative stress than exponential phase cells. A mild heat shock (37 °C, 30 min) induced thermotolerance and oxidative tolerance in both aerobic and anaerobic cells. However, prior exposure to a low concentration of H2O2 (0.1 mM, 60 min) induced protection against the lethal concentration of H2O2 but not against the lethal temperature. Sensitivity to both heat and oxidative stress was dependent on membrane lipid composition. In the case of anaerobic cells, the most stress resistant had membranes enriched in saturated fatty acids, followed in order by cells enriched in oleic and linolenic acids. Aerobic cells with membranes enriched in palmitoleic and oleic acids showed the highest resistance to stress under all conditions. In both aerobic and anaerobic cells, a mild heat shock or oxidative shock induced markedly increased levels of thiobarbituric acid reactive substance (TBARS), indicative of malondialdehyde formation and lipid damage. Anaerobic cells with membranes enriched in linolenic acid had the highest TBARS, followed by cells enriched in oleic acid, with cells enriched in saturated fatty acids showing the lowest TBARS. The results suggest that heat and oxidative stress may share a common mechanism of damage through induction of oxygen-derived free radicals, resulting in membrane lipid damage. The extent of cellular damage was related to membrane lipid composition and correlated positively with increasing unsaturation of the phospholipid fatty acyl component.

Structural and functional aspects

Abstract: Membrane Lipids and Aging Membrane-bound Ensymes Ion Channels in Biological Membranes - General Principles An-Ion Exchangers of Mammalian Cell Membranes Diversity of Transport Mechanisms in Bacteria.

Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress.

Abstract: The contribution of the unsaturation of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress was studied in a transgenic cyanobacterium. Anacystis nidulans R2-SPc was transformed with the desA gene, which encodes the Delta12-desaturase that desaturates the fatty acids of membrane lipids in Synechocystis PCC6803. The transformant acquired the ability to introduce a second double bond into palmitoleic and oleic acids. The transformation enhanced the tolerance of the photosynthetic machinery to chilling stress but it had no detectable effect on the ability to tolerate heat stress. The transformation itself did not have any effect on photosynthetic activity. These results imply that an increase in the unsaturation of membrane lipids enhances the tolerance of the photosynthetic machinery toward chilling stress but not toward heat stress and that such an increase does not affect photosynthesis within the range of physiological temperatures.

Interaction of the HIV-1 fusion peptide with phospholipid vesicles: different structural requirements for fusion and leakage

Abstract: This paper presents a study on the membrane fusion activity of a 23-residue synthetic peptide, representing the N-terminus of gp41 of the human immunodeficiency virus type I (HIV-1; LAV1a strain), in a model system involving large unilamellar vesicles (LUV) composed of the negatively charged 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). The peptide (HIVarg) induced fusion of POPG LUV as evidenced by (i) mixing of membrane lipids, (ii) mixing of aqueous vesicle contents, and (iii) an irreversible increase in vesicle size. Fusion could be induced only in the presence of millimolar concentrations of Ca2+ or Mg2+, needed for induction of vesicle aggregation; the divalent cations by themselves did not induce any fusion. The rate constant of the fusion reaction, as determined by simulation of the process according to a kinetic model, increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide was the mediator of the process. In the absence of divalent cations, the HIVarg peptide induced leakage of small molecules due to formation of pores in the membrane of single vesicles. Final extents and kinetics of this leakage process could be simulated adequately by model calculations for peptide-to-lipid ratios ranging from 1:25 to 1:750. Experiments, in which the order of peptide and Ca2+ addition to the vesicles was varied, indicated that the peptide is likely to adopt two different structures, one in the absence of Ca2+, primarily supporting leakage by formation of pores in separate vesicles, and one in the presence of Ca2+, primarily supporting fusion. Once a final structure had been established, it persisted even upon addition or removal of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

The unsaturation of membrane lipids stabilizes photosynthesis against heat stress

Abstract: The effect of the unsaturation of glycerolipids of thylakoid membranes on the heat tolerance of the photosynthetic evolution of oxygen was studied in vivo by mutation and transformation of fatty-acid desaturases in the cyanobacterium Synechocystis PCC6803. The experimental results indicate that elimination of dienoic lipid molecules decreases, to a small but distinct extent, the heat tolerance of photosynthetic oxygen evolution, but that elimination of trienoic lipid molecules has no effect on the heat tolerance. This conclusion contrasts with the previous hypothesis that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the nature of the effect of lipid unsaturation on the heat tolerance of photosynthesis.

Structural order of membranes and composition of phospholipids in fish brain cells during thermal acclimatization.

Abstract: A comparison of the structural orders of membranes of a mixed brain-cell population isolated from Cyprinus carpio L. acclimated to either summer (23-25 degrees C) or winter (5 degrees C) revealed a high degree of compensation (80%) for temperature, as assayed by electron spin resonance spectroscopy. The cells rapidly forget their thermal history and adjust the physical properties of the membranes when shifted to the other extreme of temperature either in vivo or in vitro. Phospholipids separated from both types of animals exhibit only around 10% compensation. Arachidonic and docosahexaenoic acids are the major polyunsaturated fatty acids in the brains, but the fatty acid composition of the brain total phospholipids does not vary with adaptation to temperature. Separation of phosphatidylcholines and phosphatidylethanolamines into molecular species revealed a 2- to 3-fold accumulation of 18:1/22:6, 18:1/20:4, and 18:1/18:1 species in the latter; 18:0/22:6 showed an opposite tendency. Molecular species composition of phosphatidylcholines did not vary with the temperature. The same trends of changes were seen with brains of freshwater fish from subtropical (Catla catla L.) or boreal (Acerina cernua) regions. It is concluded that the gross amount of docosahexaenoic acid (22:6) plays only a minor role in adjusting the membrane physical properties to temperature. Factors other than lipids might be involved in the adaptation processes. Due to their specific molecular architecture, molecules such as 18:1/22:6, 18:1/20:4, or 18:1/18:1 phosphatidylethanolamine might prevent the contraction of membranes in the cold and may provide an environment for some other components involved in the temperature regulation of physical properties of nerve cell membranes.

A Mutant of Arabidopsis with Increased Levels of Stearic Acid.

Abstract: A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol.

Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein

Abstract: For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein.

Cloning of omega 3 desaturase from cyanobacteria and its use in altering the degree of membrane-lipid unsaturation.

Abstract: Cyanobacteria respond to a decrease in temperature by desaturating fatty acids of membrane lipids to compensate for the decrease in membrane fluidity. Among various desaturation reactions in cyanobacteria, the desaturation of the omega 3 position of fatty acids is the most sensitive to the change in temperature. In the present study, we isolated a gene, designated desB, for the omega 3 desaturase from the cyanobacterium, Synechocystis sp. PCC 6803. The desB gene encodes a protein a 359 amino-acid residues with molecular mass of 41.9 kDa. The desB gene is transcribed as a monocistronic operon that produced a single transcript of 1.4 kb. The level of the desB transcript in cells grown at 22 degrees C was 10 times higher than that in cells grown at 34 degrees C. In order to manipulate the fatty-acid unsaturation of membrane lipids, the desB gene in Synechocystis sp. PCC 6803 was mutated by insertion of a kanamycin-resistance gene cartridge. The resultant mutant was unable to desaturate fatty acids at the omega 3 position. The desA gene, which encodes the delta 12 desaturase of Synechocystis sp. PCC 6803, and the desB gene were introduced into Synechococcus sp. PCC 7942. Whilst the parent cyanobacterium can only desaturate membrane lipids at the delta 9 position of fatty acids, the resultant transformant was able to desaturate fatty acids of membrane lipids at the delta 9, delta 12 and omega 3 positions. These results confirm the function of the desB gene and demonstrate that it is possible to genetically manipulate the fatty-acid unsaturation of membrane lipids in cyanobacteria.

Roles of lipid modifications of transducin subunits in their GDP-dependent association and membrane binding.

Abstract: Transducin is an unusually soluble and dissociable heterotrimeric G-protein, although its T alpha and T beta gamma subunits are N-acylated and farnesylated, respectively. These lipid modifications have been suggested to contribute directly to the GDP-dependent T alpha-T beta gamma association, through specific lipid recognition sites on both protein subunits. We studied the dependence of subunit association on their bound lipids and on the presence of different lipidic environments. Association of native N-acylated (nT alpha) or acyl-free recombinant (rT alpha) T alpha with farnesylated and carboxymethylated (fcT beta gamma), farnesylated (fT beta gamma), or farnesyl-free (dfT beta gamma) T beta gamma was analyzed by gradient centrifugation and gel filtration in the presence of detergent or phospholipid-cholate micelles and by cosedimentation with phospholipid vesicles. Without detergent, nT alpha GDP and fcT beta gamma associate only weakly in solution. The loss of T alpha acyl or T beta gamma farnesyl residues induces total dissociation. With detergent or lipids, isolated fcT beta gamma binds tightly to micelles or vesicles, while dfT beta gamma does not; nT alpha GDP binds weakly, while deacylated rT alpha GDP does not bind at all; and nT alpha GDP binds cooperatively with fcT beta gamma, while rT alpha GDP does not. Thus (i) the T alpha acyl chain binds weakly, whereas the T beta gamma farnesyl chain binds strongly to membrane lipids; (ii) there is no evidence for binding of the T alpha acyl chain to a polypeptide site in T beta gamma, nor for binding of the T beta gamma farnesyl chain to a polypeptidic site in T alpha, but the T alpha acyl chain seems to bind cooperatively with the T beta gamma farnesyl chain in the membrane lipids; (iii) the insertion of the two protein-attached lipids into the same membrane could contribute to the association of both subunits by favoring collision coupling of the properly oriented protein moieties on the membrane surface.

Fusogenic lipsomes and methods for making and using same

Abstract: A liposome composition is provided which contains a liposome having: (i) an outermost lipid bilayer containing, in addition to a neutral, bilayer preferring lipid, a fusion-promoting effective amount of an ionizable lipid having a protonatable, cationic headgroup and an unsaturated acyl chain; and (ii) a compartment adjacent to the outermost lipid bilayer which contains an aqueous solution having a first pH. External to the liposome in the composition is an aqueous solution having a second pH. The first pH is less than the pK a of the ionizable lipid in the outermost lipid bilayer and the second pH is greater than the pK a of the ionizable lipid in the outermost lipid bilayer, such that there is a pH gradient across the outermost lipid bilayer and the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer in response to the gradient. The liposome can be fused in a controlled manner to another lipid bilayer, for example, the plasma membrane of a mammalian cell, by degrading the pH gradient when fusion is to occur, such that the ionizable lipid is neutral, evenly distributed in the outermost lipid bilayer and, fusogenic. Controlled fusion can be used to control the delivery of a biologically active agent entrapped in the liposome into a cell.

Radiolabeling studies of lipids and fatty acids in nannochloropsis (eustigmatophyceae), an oleaginous marine alga1

Abstract: The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [14C]-bicarbonate or [14C]-acetate. [14C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [14C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [14C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C20 fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C18 fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C20 fatty acids.

Free radical metabolism, pigment degradation and lipid peroxidation in leaves during senescence

Abstract: Various lines of evidence suggest that the controlled increase in the production of oxygen radicals is an important factor involved in phytohormone metabolism, selective changes in membrane permeability, degradation of photosynthetic pigments and unsaturated membrane lipids, oxidative modification and subsequent proteolytic degradation, and other events occurring in ageing and senescing leaves. Increased lipid peroxidation, appearance of chlorophyll allomerised products, as well as fluorescent ‘lipofuscin-like’ pigments have been documented in different systems of leaf senescence. On the other hand, the accumulation of peroxidised products may result from the alteration to native structural organisation and energy dissipation reactions of the photosynthetic apparatus. Although there are contradictory and incomplete data on the activity of the systems involved in the metabolism of oxygen radicals, it seems that senescing leaves retain, at least in part, their defence potential against both activated oxygen species and toxic lipid peroxidation products. This provides the plant with the ability to successfully dismantle its photosynthetic apparatus during senescence in a relatively safe manner.

Annexin IV reduces the rate of lateral lipid diffusion and changes the fluid phase structure of the lipid bilayer when it binds to negatively charged membranes in the presence of calcium.

Abstract: Bovine annexin IV (endonexin) was bound to supported planar bilayers composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) in the first monolayer facing the substrate, and varying mole fractions of POPC, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and small amounts of the fluorescent lipid analogs NBD-PC or NBD-PG in the second monolayer facing the large aqueous compartment. Lateral diffusion coefficients and mobile fractions of these phospholipids were measured by fluorescence recovery after photobleaching (FRAP) as a function of protein concentration and lipid composition in the presence of 2 mM CaCl2 or 1 mM EDTA. In the absence of annexin IV, the lateral diffusion coefficients depended only little on the POPC:POPG ratios and were approximately 3.0 microns2/s for NBD-PG (no Ca2+), 2.5 microns2/s for NBD-PG (2 mM Ca2+), and 1.6 microns2/s for NBD-PC (with or without 2 mM Ca2+). In the presence of 2 mM Ca2+ these diffusion coefficients decreased as a function of the added annexin concentration. A transition from a state with "rapid" lipid diffusion to a state with "slow" lipid diffusion occurred at about 80 nM annexin IV and was independent of the POPC:POPG ratio. In addition to reducing the lipid lateral diffusion coefficients, annexin IV also gave rise to two-component lateral diffusion of the lipids in these mixed bilayers. The split of the single diffusion coefficient of NBD-PG into two components occurred at most POPC:POPG ratios upon binding of annexin IV, but required higher annexin concentrations at mole fractions of POPC between 66 and 82 mol % than at high mole fractions of POPG or 90 mol % POPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of fluorescent lipid peroxidation products

Abstract: Publisher Summary Lipid hydroperoxides decompose into many secondary products including aldehydes, such as malonaldehyde (MA), which is a possible precursor for the fluorescent lipid peroxidation products formed in a variety of systems. The formation of age pigments (lipofuscin and ceroid pigments) is accompanied by the appearance of high molecular weight proteins or polymeric lipid-protein complexes, and the substances have been characterized by several investigators as having a strong fluorescence with a maximum in the region of 430–490 nm on excitation with ultraviolet light. The pigments are probably composed of heterogeneous complexes including lipid-soluble and lipid-insoluble fluorescent lipid peroxidation products. Scientific interest in fluorescent lipid peroxidation products in biological systems is rapidly developing, because it seems likely that investigation of fluorescent lipid peroxidation products leads to a clearer understanding of in vivo lipid peroxidation. This chapter introduces methods for the extraction and spectrophotofluorometric analyses of fluorescent lipid peroxidation products formed in both in vitro and in vivo systems.

Cholesterol sulfate, a novel activator for the eta isoform of protein kinase C.

Abstract: Activity of protein kinase C depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. In the present study, we demonstrated a novel activation mechanism of the eta isoform of protein kinase C (nPKC eta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that the nPKC eta was activated by cholesterol sulfate, a metabolite of cholesterol formed during squamous differentiation. This activation was greater than that by phosphatidylserine plus phorbol ester; the Vmax for the activation by cholesterol sulfate was 3.6 times that by phosphatidylserine plus phorbol ester, while Kms were almost equal. In the presence of cholesterol sulfate, phorbol ester only weakly enhanced the activity of nPKC eta. Activation of nPKC eta by cholesterol sulfate was further demonstrated by autophosphorylation of the kinase molecule. However, the alpha and delta isoforms of protein kinase C were not activated by cholesterol sulfate. The present observation affords a new insight into a signal transduction pathway of squamous differentiation.