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Sample records for phospholipid model membranes

  1. Patterning and characterization of model phospholipid membranes

    NASA Astrophysics Data System (ADS)

    Kassu, Aschalew; Calzzani, Fernando A., Jr.; Taguenang, Jean M.; Sileshi, Redahegn K.; Sharma, Anup

    2008-08-01

    Phospholipid, which is a building block of biological membranes, plays an important role in compartmentalization of cellular reaction environment and control of the physicochemical conditions inside the reaction environment. Phospholipid bilayer membrane has been proposed as a natural biocompatible platform for attaching biological molecules like proteins for biosensing related application. Due to the enormous potential applications of biomimetic model biomembranes, various techniques for depositions and patterning of these membranes onto solid supports and their possible biotechnological applications have been reported by different groups. In this work, patterning of phospholipid thin-films is accomplished by interferometric lithography as well as using lithographic masks in liquid phase. Surface Enhanced Raman Spectroscopy and Atomic Force microscopy are used to characterize the model phospholipid membrane and the patterning technique. We describe an easy and reproducible technique for direct patterning of azo-dye (NBD)-labeled phospholipid (phosphatidylcholine) in aqueous medium using a low-intensity 488 nm Ar+ laser and various kinds of lithographic masks.

  2. Biophysical studies of cholesterol in unsaturated phospholipid model membranes

    NASA Astrophysics Data System (ADS)

    Williams, Justin Adam

    Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distributed to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. This research employs model membranes of well-defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega-3 (n-3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids. The molecular organization of 1-[2H31]palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC-d31) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31) in membranes with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid-state 2H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n-3 PUFAs found in fish oil, while PEPC-d31 and PDPC-d31 are phospholipids containing the respective PUFAs at the sn-2 position and a perdeuterated palmitic acid at the sn-1 position. Analysis of spectra recorded as a function of temperature indicates that in both cases, formation of PUFA-rich (less ordered) and SM-rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infiltrate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%). The implication is that n-3

  3. A new PAMPA model proposed on the basis of a synthetic phospholipid membrane.

    PubMed

    Yu, Hui; Wang, Qi; Sun, Ying; Shen, Ming; Li, He; Duan, Yourong

    2015-01-01

    The purpose of this work was to investigate the synthetic phospholipid dependence of permeability measured by parallel artificial membrane permeability assay (PAMPA) method. Three phospholipids with hydrophobic groups of different lengths and phosphorylcholine as the hydrophilic group were concisely synthesized. Ten model drug molecules were selected because of their distinct human fraction absorbed (%FA) values and various pKa characteristics. In vitro drug permeation experiments were designed to determine the effect of the incubation time (4-20 h), pH gradient (4.6-9.32) and carbon chain length (8, 10, 12) on the drug permeability through the synthetic phospholipid membrane in the PAMPA system. The results showed that intensive and significant synthetic phospholipids dependence of permeability influenced by the length of lipid's hydrophobic carbon chain. The effective permeability constant (Pe) of each drug increased rapidly with time, then decreased slightly after reaching the maximum; the pH gradient changed the drug permeability according to the pH-partition hypothesis for drugs with diverse pKa values; and longer hydrophobic chains in the synthetic phospholipid membrane improved the drug permeability, as observed for all test drugs at almost all incubation time points. This newly proposed PAMPA model considered the synthetic phospholipid membrane and showed good Pe-%FA correlation for the passive transport of drugs, making it a helpful supplementary method for PAMPA systems. PMID:25647086

  4. A method for the modulation of membrane fluidity: homogeneous catalytic hydrogenation of phospholipids and phospholipids and phospholipid-water model biomembranes.

    PubMed Central

    Chapman, D; Quinn, P J

    1976-01-01

    The fatty acids associated with phospholipids of cell membranes, and particularly their degree of unsaturation, contribute to the fluidity of their structure and hance determine many of their biological properties. We describe a technique for modulating membrane fluidity which consists of hydrogenating the unsaturated double bonds of membrane phospholipids. This has been accomplished using a homogeneous catalyst. The process has been applied to phospholipids in organic solvents, to phospholipids dispersed as multibilayers in aqueous systems, and also to sonicated preparations of phospholipids arranged as single bilayer vesicles. Preliminary experiments have also been performed with biological membranes. These results indicate that the process of homogeneous catalysis for the modulation of lipid fluidity of biological cell membranes may have considerable future biological and biochemical application. PMID:1069280

  5. New insights into water-phospholipid model membrane interactions.

    PubMed

    Milhaud, Jeannine

    2004-05-27

    Modulating the relative humidity (RH) of the ambient gas phase of a phospholipid/water sample for modifying the activity of phospholipid-sorbed water [humidity-controlled osmotic stress methods, J. Chem. Phys. 92 (1990) 4519 and J. Phys. Chem. 96 (1992) 446] has opened a new field of research of paramount importance. New types of phase transitions, occurring at specific values of this activity, have been then disclosed. Hence, it is become recognized that this activity, like the temperature T, is an intensive parameter of the thermodynamical state of these samples. This state can be therefore changed (phase transition) either, by modulating T at a given water activity (a given hydration level), or, by modulating the water activity, at a given T. The underlying mechanisms of these two types of transition differ, especially when they appear as disorderings of fatty chains. In lyotropic transitions, this disordering follows from two thermodynamical laws. First, acting on the activity (the chemical potential) of water external to a phospholipid/water sample, a transbilayer gradient of water chemical potential is created, leading to a transbilayer flux of water (Fick's law). Second, water molecules present within the hydrocarbon region of this phospholipid bilayer interact with phospholipid molecules through their chemical potential (Gibbs-Duhem relation): the conformational state of fatty chains (the thermodynamical state of the phospholipid molecules) changes. This process is slow, as revealed by osmotic stress time-resolved experiments. In thermal chain-melting transitions, the first rapid step is the disordering of fatty chains of a fraction of phospholipid molecules. It occurs a few degrees before the main transition temperature, T(m), during the pretransition and the sub-main transition. The second step, less rapid, is the redistribution of water molecules between the different parts of the sample, as revealed by T-jump time-resolved experiments. Finally, in

  6. Phospholipid interactions in model membrane systems. I. Experiments on monolayers.

    PubMed Central

    Mingins, J; Stigter, D; Dill, K A

    1992-01-01

    We study the lateral headgroup interactions among phosphatidylcholine (PC) molecules and among phosphatidylethanolamine (PE) molecules in monolayers and extend our previous models. In this paper, we present an extensive set of pressure-area isotherms and surface potential experiments on monolayers of phospholipids ranging from 14 to 22 carbons in length at the n-heptane/water interface, over a wide range of temperature, salt concentration, and pH on the acid side. The pressure data presented here are a considerable extension of previous data (1) to higher surface densities, comprehensively checked for monolayer loss, and include new data on PE molecules. We explore surface densities ranging from extremely low to intermediate, near to the main phase transition, in which range the surface pressures and potentials are found to be independent of the chain length. Thus, these data bear directly on the headgroup interactions. These interactions are observed to be independent of ionic strength. PC and PE molecules differ strongly in two respects: (a) the lateral repulsion among PC molecules is much stronger than for PE, and (b) the lateral repulsion among PC molecules increases strongly with temperature whereas PE interactions are almost independent of temperature. Similarly, the surface potential for PC is found to increase with temperature whereas for PE it does not. In this and the following paper we show that these data from dilute to semidilute monolayers are consistent with a theoretical model that predicts that, independent of coverage, for PC the P-N+ dipole is oriented slightly into the oil phase because of the hydrophobicity of the methyl groups, increasingly so with temperature, whereas for PE the P-N+ dipole is directed into the water phase. PMID:1617140

  7. Phospholipid Diffusion Coefficients of Cushioned Model Membranes determined via Z-Scan Fluorescence Correlation Spectroscopy

    PubMed Central

    Sterling, Sarah M.; Allgeyer, Edward S.; Fick, Jörg; Prudovsky, Igor; Mason, Michael D.; Neivandt, David J.

    2013-01-01

    Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins, or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir Schaefer method on a hydrogel layer is potentially an effective mimic of the cross-section of a biological membrane, and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and coworkers, revealed that phospholipid diffusion changes from raft-like to free diffusion as the temperature is increased; an insight into the dynamic behavior of hydrogel supported membranes not previously reported. PMID:23705855

  8. Studies on the interactions of bisphenols with anionic phospholipids of decomposer membranes in model systems.

    PubMed

    Broniatowski, Marcin; Sobolewska, Katarzyna; Flasiński, Michał; Wydro, Paweł

    2016-04-01

    Bisphenol A (BPA) and other bisphenols constitute a class of organic pollutants, which because of their estrogenic properties, low dose activity and bioaccumulation pose considerable risk for public health as well as for the environment. Accumulated in the sediment bisphenols can endanger the decomposers' populations being incorporated into their cellular membranes; however, the mechanism of their membrane activity is unknown. Therefore, to study these phenomena we applied anionic phospholipid Langmuir monolayers as simple but versatile models of decomposers biomembranes. Phosphatidylglycerols and cardiolipins are not only the main components of bacterial membranes but also of crucial importance in mitochondrial and thylakoid membranes in eukaryotic cells. In our investigations we applied five compounds of the bisphenol class most commonly detected in the environment. To characterize the bisphenols-model membrane interactions we applied multiple mutually independent methods of physical chemistry; namely: the Langmuir monolayer technique, surface potential measurements, Brewster angle microscopy for the visualization of the monolayers' texture and grazing incidence X-ray diffraction for the discussion of the phospholipids packing within the monolayers. Our studies indicated that all the investigated bisphenols interact with the model membrane, but the strength of the interactions is dependent on the bisphenol structure and hydrophobicity and the fluidity of the model membranes. We proved that bisphenol S often treated as the least toxic BPA analog can also be incorporated to the model membranes changing their structure and fluidity. PMID:26806160

  9. Effects of structure on the interactions between five natural antimicrobial compounds and phospholipids of bacterial cell membrane on model monolayers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be...

  10. Number of free hydroxyl groups on bile acid phospholipids determines the fluidity and hydration of model membranes.

    PubMed

    Sreekanth, Vedagopuram; Bajaj, Avinash

    2013-10-10

    Interactions of synthetic phospholipids with model membranes determines the drug release capabilities of phospholipid vesicles at diseased sites. We performed 1,6-diphenyl-1,3,5-hexatriene (DPH)-based fluorescence anisotropy, Laurdan-based membrane hydration, and differential scanning calorimetry (DSC) studies to cognize the interactions of three bile acid phospholipids, lithocholic acid-phosphocholine (LCA-PC), deoxycholic acid-phosphocholine (DCA-PC), and cholic acid-phosphocholine (CA-PC) with model membranes. These studies revealed that bile acid phospholipids increases membrane fluidity in DCA-PC > CA-PC > LCA-PC order, indicating that induction of membrane fluidity is contingent on the number and positioning of free hydroxyl groups on bile acids. Similarly, DCA-PC causes maximum membrane perturbations due to the presence of a free hydroxyl group, whereas LCA-PC induces gel phase in membranes due to hydrophobic bile acid acyl chain interactions. These DCA-PC-induced membrane perturbations induce a drastic decrease in phase transition temperature (Tm) as determined by calorimetric studies, whereas doping of LCA-PC causes phase transition broadening without change in Tm. Doping of CA-PC induces membrane perturbations and membrane hydration like DCA-PC but sharpening of phase transition at higher doping suggests self-association of CA-PC molecules. Therefore these differential mode of interactions between bile acid phospholipids and model membranes would help in the future for their use in drug delivery. PMID:24079709

  11. Physiochemical Properties of Aluminum Adjuvants Elicit Differing Reorganization of Phospholipid Domains in Model Membranes.

    PubMed

    Antúnez, Lorena R; Livingston, Andrea; Berkland, Cory; Dhar, Prajnaparamita

    2016-05-01

    Most vaccines contain aluminum adjuvants; however, their exact mechanism of action remains unclear. A novel mechanism by Shi and colleagues proposes aluminum adjuvants may enhance immune activation by binding and reorganizing lipids that are key components of lipid rafts. To better understand the specificity of interaction between aluminum adjuvants and the cell membrane lipids, we present a biophysical study of lipid domain clustering in simple model phospholipid monolayers containing dipalmitoyl-phosphatidylcholine (DPPC) and dioleoyl-phosphatidylcholine (DOPC) exposed to two aluminum adjuvants, Alhydrogel and Adju-Phos. Surface pressure measurements and fluorescence microscopy images verified aluminum adjuvant-induced increase in lipid domain size, even in the key lipid raft components. Additionally, adjuvant induced lipid clustering differed based on the physicochemical properties of the adjuvants. Alhydrogel appeared to reduce monolayer compressibility and insert into the monolayer, while Adju-Phos induced more significant changes in domain size, without compromising the integrity of the monolayer. The Alhydrogel and Adju-Phos-mediated reorganization of phospholipid domains reported here supports the new mechanistic paradigm proposed by Shi and co-workers, and further suggests that lipid clustering is induced even in simple phospholipid membranes. The results present the basis for future exploration into lipid-mediated mechanisms of action for adjuvants. PMID:26998680

  12. Effects of verbascoside, a phenylpropanoid glycoside from lemon verbena, on phospholipid model membranes.

    PubMed

    Funes, Lorena; Laporta, Olga; Cerdán-Calero, Manuela; Micol, Vicente

    2010-02-01

    Phenylpropanoid glycosides are water-soluble compounds widely distributed, most of them deriving from medicinal herbs. Among them, verbascoside or acteoside has exhibited a wide biological activity, being free radical scavenging the most representative one. Moreover, antitumor, antimicrobial, anti-inflammatory, anti-thrombotic and wound healing properties have been previously described. Herein, the interaction of verbascoside with phospholipid membranes has been studied by means of differential scanning calorimetry, fluorescence anisotropy and dynamic light scattering. Verbascoside showed stronger affinity for negatively charged membranes composed of phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This compound promoted phase separation of lipid domains in PC membranes and formed a stable lipid complex with and approximate phospholipid/verbascoside ratio of 4:1. Despite its hydrophilic character, verbascoside's caffeoyl moiety was located deep into the hydrophobic core of PC membranes and was almost inaccessible to spin probes located at different depths in PG membranes. This compound affected the ionization behavior of the PG phosphate group and most likely interacted with the vesicles surface. The presence of verbascoside decreased the particle size in PG unilamellar vesicles through the increase of the phospholipid head group area. A localization of verbascoside filling the upper region of PG bilayers close to the phospholipid/water interface is proposed. These effects on membranes may help to understand the mechanism of the biological activity of verbascoside and other similar phenylpropanoid glycosides. PMID:19925782

  13. Adsorption of ruthenium red to phospholipid membranes.

    PubMed Central

    Voelker, D; Smejtek, P

    1996-01-01

    We have measured the distribution of the hexavalent ruthenium red cation (RuR) between water and phospholipid membranes, have shown the critical importance of membrane negative surface charge for RuR binding, and determined the association constant of RuR for different phospholipid bilayers. The studies were performed with liposomes made of mixtures of zwitterionic L-alpha-phosphatidylcholine (PC), and one of the negatively charged phospholipids: L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylinositol (PI), or L-alpha-phosphatidylglycerol (PG). Lipid composition of PC:PX membranes was 1:0, 19:1, 9:1, and 4:1. Liposomes were processed using freeze-and-thaw treatment, and their size distribution was characterized by light scattering and electron microscopy. Experimental distribution isotherms of RuR obtained by ultracentrifugation and spectrophotometry can be reproduced with the Langmuir-Stern-Grahame model, assuming that RuR behaves in the diffuse double layer as an ion with effective valency < 6. In terms of this model, PC-PS, PC-PI, and PC-PG membranes were found to be electrostatically equivalent and the intrinsic association constants of RuR were obtained. RuR has highest affinity to PS-containing membranes; its association constant for PC-PI and PC-PG membranes is about 5 times smaller than that for PC-PS membranes. From the comparison of RuR binding to mixed negatively charged phospholipid membranes and RuR binding to sarcoplasmic reticulum (SR), we conclude that the low-affinity RuR binding sites may indeed be associated with the lipid bilayer of SR. PMID:8789099

  14. Supported phospholipid/alkanethiol biomimetic membranes: insulating properties.

    PubMed Central

    Plant, A L; Gueguetchkeri, M; Yap, W

    1994-01-01

    A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes. PMID:7811924

  15. Quantum dot effects upon the interaction between porphyrins and phospholipids in cell membrane models.

    PubMed

    Parra, Gustavo G; Borissevitch, Galina; Borissevitch, Iouri; Ramos, Ana P

    2016-04-01

    This study employed surface pressure isotherms and spectroscopic techniques to investigate the effect of quantum dots on the interaction between porphyrins and phospholipids using Langmuir monolayers and Langmuir-Blodgett films formed from negatively charged DMPA (the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid) and zwitterionic DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) as cell membrane models in the presence of 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin (TMPyP), 5,10,15,20-tetrakis(p-sulfonato-phenyl) porphyrin (TPPS4) and PEG-coated CdSe/ZnS quantum dots (QD). The porphyrins present at the monolayer subphase affected the organization of the lipids at the air/liquid interface, as shown by the changes in the surface pressure-surface area isotherms. QDs enhanced the interaction of TMPyP with DMPA, improving their transference from the liquid monolayers to solid supports. A higher amount of TMPyP was transferred to DMPA-Langmuir-Blodgett films when the QDs were present in the subphase as evidenced by the UV-Vis data. For DPPC the surface effects due to the presence of QDs are less evident. PMID:26518694

  16. Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system.

    PubMed

    Prosser, R S; Hwang, J S; Vold, R R

    1998-05-01

    A stable smectic phospholipid bilayer phase aligned with the director parallel to the magnetic field can be generated by the addition of certain trivalent paramagnetic lanthanide ions to a bicellar solution of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in water. Suitable lanthanide ions are those with positive anisotropy of their magnetic susceptibility, namely Eu3+, Er3+, Tm3+, and Yb3+. For samples doped with Tm3+, this phase extends over a wide range of Tm3+ concentrations (6-40 mM) and temperatures (35-90 degrees C) and appears to undergo a transition from a fluid nematic discotic to a fluid, but highly ordered, smectic phase at a temperature that depends on the thulium concentration. As a membrane mimetic, these new, positively ordered phospholipid phases have high potential for structural studies using a variety of techniques such as magnetic resonance (EMR and NMR), small-angle x-ray and neutron diffraction, as well as optical and infrared spectroscopy. PMID:9591667

  17. Nonenzymatic Reactions above Phospholipid Surfaces of Biological Membranes: Reactivity of Phospholipids and Their Oxidation Derivatives

    PubMed Central

    Solís-Calero, Christian; Ortega-Castro, Joaquín; Frau, Juan; Muñoz, Francisco

    2015-01-01

    Phospholipids play multiple and essential roles in cells, as components of biological membranes. Although phospholipid bilayers provide the supporting matrix and surface for many enzymatic reactions, their inherent reactivity and possible catalytic role have not been highlighted. As other biomolecules, phospholipids are frequent targets of nonenzymatic modifications by reactive substances including oxidants and glycating agents which conduct to the formation of advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs). There are some theoretical studies about the mechanisms of reactions related to these processes on phosphatidylethanolamine surfaces, which hypothesize that cell membrane phospholipids surface environment could enhance some reactions through a catalyst effect. On the other hand, the phospholipid bilayers are susceptible to oxidative damage by oxidant agents as reactive oxygen species (ROS). Molecular dynamics simulations performed on phospholipid bilayers models, which include modified phospholipids by these reactions and subsequent reactions that conduct to formation of ALEs and AGEs, have revealed changes in the molecular interactions and biophysical properties of these bilayers as consequence of these reactions. Then, more studies are desirable which could correlate the biophysics of modified phospholipids with metabolism in processes such as aging and diseases such as diabetes, atherosclerosis, and Alzheimer's disease. PMID:25977746

  18. Bolaamphiphiles Promote Phospholipid Translocation Across Vesicle Membranes

    PubMed Central

    Forbes, Christopher C.; DiVittorio, Kristy M.; Smith, Bradley D.

    2008-01-01

    A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end-groups connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their abilities to affect the dynamics of a surrounding bilayer membrane. The goal was to determine if the bolaamphiphiles promote the translocation of phospholipids across vesicle membranes. The bolaamphiphiles were incorporated at low levels (up to 5 mol%) in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Inward translocation assays were performed using fluorescent, NBD-labeled phospholipid probes with phosphocholine (PC) or phosphoglycerol (PG) head-groups. The membrane-spanning bolaamphiphiles promote the translocation of both phospholipid probes in the order PG > PC, while shorter bolaamphiphiles (structures that must adopt a U-shape and keep both end-groups in the same leaflet of the membrane), and regular amphiphiles with one hydrophilic end-group, are inactive. These results are an exception to the rule-of-thumb that membrane-spanning bolaamphiphiles are inherently membrane stabilizing molecules that inhibit all types of membrane transport. PMID:16834395

  19. Contribution of the Tyr-1 in Plantaricin149a to Disrupt Phospholipid Model Membranes

    PubMed Central

    Lopes, José L. S.; Gómara, Maria J.; Haro, Isabel; Tonarelli, Georgina; Beltramini, Leila M.

    2013-01-01

    Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from −17.2 kcal/mol to −10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface. PMID:23749115

  20. Nature of the charged headgroup determines the fusogenic potential and membrane properties of lithocholic acid phospholipids.

    PubMed

    Bhargava, Priyanshu; Singh, Manish; Sreekanth, Vedagopuram; Bajaj, Avinash

    2014-08-01

    Phospholipids play a crucial role in many cellular processes ranging from selective membrane permeability, to membrane fission and fusion, to cellular signaling. Headgroups of phospholipids determine the membrane properties and fusogenicity of these lipids with target cell membranes. We studied the fusogenic and membrane properties of phospholipids possessing unnatural charged headgroups with model membranes using laurdan based membrane hydration studies, DPH based membrane fluidity, and differential scanning calorimetry. We unravel that fusogenicity, membrane hydration, and fluidity of membranes are strongly contingent on the nature of the phospholipid charged headgroup. Our studies unraveled that introduction of bulky headgroups like dimethylamino pyridine induces maximum membrane hydration and perturbations with high fusogenicity as compared to small headgroup based phospholipids. These phospholipids also have the capability of high retention in DPPC membranes. Hydration and fluidity of these phospholipid-doped DPPC membranes are contingent on the nature of the charged headgroup. This study would help in future design of phospholipid based nanomaterials for effective drug delivery. PMID:25029367

  1. Self-reproducing catalyst drives repeated phospholipid synthesis and membrane growth

    PubMed Central

    Hardy, Michael D.; Yang, Jun; Selimkhanov, Jangir; Cole, Christian M.; Tsimring, Lev S.; Devaraj, Neal K.

    2015-01-01

    Cell membranes are dynamic structures found in all living organisms. There have been numerous constructs that model phospholipid membranes. However, unlike natural membranes, these biomimetic systems cannot sustain growth owing to an inability to replenish phospholipid-synthesizing catalysts. Here we report on the design and synthesis of artificial membranes embedded with synthetic, self-reproducing catalysts capable of perpetuating phospholipid bilayer formation. Replacing the complex biochemical pathways used in nature with an autocatalyst that also drives lipid synthesis leads to the continual formation of triazole phospholipids and membrane-bound oligotriazole catalysts from simpler starting materials. In addition to continual phospholipid synthesis and vesicle growth, the synthetic membranes are capable of remodeling their physical composition in response to changes in the environment by preferentially incorporating specific precursors. These results demonstrate that complex membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks. PMID:26100914

  2. Synaptogenesis: Modulation by Availability of Membrane Phospholipid Precursors.

    PubMed

    Cansev, Mehmet

    2016-09-01

    Phospholipids are the main constituents of brain membranes. Formation of new membranes requires that uridine, the omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), and choline, the three circulating precursors of major phospholipids, interact via the Kennedy pathway. Supplementation of laboratory rodents with uridine, DHA and choline enhances the amount of brain membranes as well as synaptic proteins and increases the number of dendritic spines, the essential cytological precursor of new synapses. Hence, the newly formed membranes are utilized for synaptogenesis which underlies increased synaptic functioning evidenced by enhanced neurotransmission and cognition. In addition, this supplementation ameliorates the degeneration in a rat model of Parkinson's disease and mouse models of Alzheimer's disease (AD) when used in combination with several vitamins and cofactors. Hence, accumulating evidence shows that increasing the availability of phospholipid precursors, vitamins and cofactors to the brain through dietary supplementation enhances the formation of new synapses and provides protection under neurodegenerative conditions. The combination has been tested in clinical trials and a medication has been marketed for early-stage AD patients. PMID:27250850

  3. Water at the Surfaces of Aligned Phospholipid Multi-Bilayer Model Membranes Probed with Ultrafast Vibrational Spectroscopy

    PubMed Central

    Zhao, Wei; Moilanen, David E.; Fenn, Emily E.; Fayer, Michael D.

    2009-01-01

    The dynamics of water at the surface of artificial membranes composed of aligned multibilayers of the phospholipid dilauroyl phosphatidylcholine (DLPC) are probed with ultrafast polarization selective vibrational pump-probe spectroscopy. The experiments are performed at various hydration levels, x = 2 – 16 water molecules per lipid at 37 °C. The water molecules are ~1 nm above or below the membrane surface. The experiments are conducted on the OD stretching mode of dilute HOD in H2O to eliminate vibrational excitation transfer. The FT-IR absorption spectra of the OD stretch in the DLPC bilayer system at low hydration levels shows a red-shift in frequency relative to bulk water, which is in contrast to the blue shift often observed in systems such as water nanopools in reverse micelles. The spectra for x = 4 – 16 can be reproduced by a superposition of the spectra for x = 2 and bulk water. IR Pump-probe measurements reveal that the vibrational population decays (lifetimes) become longer as the hydration level is decreased. The population decays are fit well by biexponential functions. The population decays, measured as a function of the OD stretch frequency, suggest the existence of two major types of water molecules in the interfacial region of the lipid bilayers. One component may be a clathrate-like water cluster near the hydrophobic choline group and the other may be related to the hydration water molecules mainly associated with the phosphate group. As the hydration level increases, the vibrational lifetimes of these two components decrease, suggesting a continuous evolution of the hydration structures in the two components associated with the swelling of the bilayers. The agreement of the magnitudes of the two components obtained from IR spectra with those from vibrational lifetime measurements further supports the two component model. The vibrational population decay fitting also gives an estimation of the number of phosphate-associated water molecules

  4. Looking Beyond Structure: Membrane Phospholipids of Skeletal Muscle Mitochondria.

    PubMed

    Heden, Timothy D; Neufer, P Darrell; Funai, Katsuhiko

    2016-08-01

    Skeletal muscle mitochondria are highly dynamic and are capable of tremendous expansion to meet cellular energetic demands. Such proliferation in mitochondrial mass requires a synchronized supply of enzymes and structural phospholipids. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are generated in skeletal muscle. Herein we describe how each class of phospholipids that constitute mitochondrial membranes are synthesized and/or imported, and summarize genetic evidence indicating that membrane phospholipid composition represents a significant modulator of skeletal muscle mitochondrial respiratory function. We also discuss how skeletal muscle mitochondrial phospholipids may mediate the effect of diet and exercise on oxidative metabolism. PMID:27370525

  5. Enhancement by cytidine of membrane phospholipid synthesis

    NASA Technical Reports Server (NTRS)

    G-Coviella, I. L.; Wurtman, R. J.

    1992-01-01

    Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.

  6. Influence of lipids with hydroxyl-containing head groups on Fe2+ (Cu2+)/H2O2-mediated transformation of phospholipids in model membranes.

    PubMed

    Olshyk, Viktoriya N; Melsitova, Inna V; Yurkova, Irina L

    2014-01-01

    Under condition of ROS formation in lipid membranes, free radical reactions can proceed in both hydrophobic (peroxidation of lipids, POL) and polar (free radical fragmentation) parts of the bilayer. Free-radical fragmentation is typical for the lipids containing a hydroxyl group in β-position with respect to an ester or amide bond. The present study has been undertaken to investigate free-radical transformations of phospholipids in model membranes containing lipids able to undergo fragmentation in their polar part. Liposomes from egg yolk lecithin containing saturated or monounsaturated glycero- and sphingolipids were subjected to the action of an HO* - generating system - Fe(2+)(Cu(2+))/H2O2/Asc, and the POL products were investigated. In parallel with this, the effects of monoacylglycerols and scavengers of reactive species on Fe(2+)(Cu(2+))/H2O2/Asc - mediated free-radical fragmentation of phosphatidylglycerols were studied. Hydroxyl-containing sphingolipids and glycerolipids, which undergo free-radical fragmentation under such conditions, manifested antioxidant properties in the model membranes. In the absence of HO groups in the lipid structure, the effect was either pro-oxidant or neutral. Monoacylglycerols slowed down the rate of both peroxidation in the hydrophobic part and free-radical fragmentation in the hydrophilic part of phospholipid membrane. Scavengers of reactive species inhibited the fragmentation of phosphatidylglycerol substantially. Thus, the ability of hydroxyl-containing lipids to undergo free-radical fragmentation in polar part apparently makes a substantial contribution to the mechanism of their protector action. PMID:24189590

  7. Surface acoustic wave biosensor as a tool to study the interaction of antimicrobial peptides with phospholipid and lipopolysaccharide model membranes.

    PubMed

    Andrä, Jörg; Böhling, Arne; Gronewold, Thomas M A; Schlecht, Ulrich; Perpeet, Markus; Gutsmann, Thomas

    2008-08-19

    Surface acoustic wave biosensors are a powerful tool for the study of biomolecular interactions. The modulation of a surface-confined acoustic wave is utilized here for the analysis of surface binding. Phase and amplitude of the wave correspond roughly to mass loading and viscoelastic properties of the surface, respectively. We established a procedure to reconstitute phospholipid and lipopolysaccharide bilayers on the surface of a modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure included the formation of a self-assembled monolayer of 11-mercaptoundecanol, covalent coupling of carboxymethyl-dextran, and subsequent coating with a poly- l-lysine layer. The lipid coverage of the surface is highly reproducible and homogeneous as demonstrated in atomic force micrographs. Ethanol/triton treatment removed the lipids completely, which provided the basis for continuous sequences of independent experiments. The setup was applied to investigate the binding of human cathelicidin-derived peptide LL32, as an example for antimicrobial peptides, to immobilized phosphatidylserine membranes. The peptide-membrane interaction results in a positive phase shift and an increase in amplitude, indicating a mass increase along with a loss in viscosity. This suggests that the bilayer becomes more rigid upon interaction with LL32. PMID:18605705

  8. Phospholipid Composition of Membranes Directs Prions Down Alternative Aggregation Pathways

    PubMed Central

    Robinson, Philip J.; Pinheiro, Teresa J.T.

    2010-01-01

    Abstract Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in β-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth. PMID:20409471

  9. Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane

    PubMed Central

    Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C.; Fradin, Cécile

    2015-01-01

    Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol’s condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content. PMID:26529029

  10. Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes.

    PubMed

    Pankov, R; Markovska, T; Antonov, P; Ivanova, L; Momchilova, A

    2006-09-01

    Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers. PMID:17197729

  11. Correlation of insulin-enhancing properties of vanadium-dipicolinate complexes in model membrane systems: phospholipid langmuir monolayers and AOT reverse micelles.

    PubMed

    Sostarecz, Audra G; Gaidamauskas, Ernestas; Distin, Steve; Bonetti, Sandra J; Levinger, Nancy E; Crans, Debbie C

    2014-04-22

    We explore the interactions of V(III) -, V(IV) -, and V(V) -2,6-pyridinedicarboxylic acid (dipic) complexes with model membrane systems and whether these interactions correlate with the blood-glucose-lowering effects of these compounds on STZ-induced diabetic rats. Two model systems, dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers and AOT (sodium bis(2-ethylhexyl)sulfosuccinate) reverse micelles present controlled environments for the systematic study of these vanadium complexes interacting with self-assembled lipids. Results from the Langmuir monolayer studies show that vanadium complexes in all three oxidation states interact with the DPPC monolayer; the V(III) -phospholipid interactions result in a slight decrease in DPPC molecular area, whereas V(IV) and V(V) -phospholipid interactions appear to increase the DPPC molecular area, an observation consistent with penetration into the interface of this complex. Investigations also examined the interactions of V(III) - and V(IV) -dipic complexes with polar interfaces in AOT reverse micelles. Electron paramagnetic resonance spectroscopic studies of V(IV) complexes in reverse micelles indicate that the neutral and smaller 1:1 V(IV) -dipic complex penetrates the interface, whereas the larger 1:2 V(IV) complex does not. UV/Vis spectroscopy studies of the anionic V(III) -dipic complex show only minor interactions. These results are in contrast to behavior of the V(V) -dipic complex, [VO2 (dipic)](-) , which penetrates the AOT/isooctane reverse micellar interface. These model membrane studies indicate that V(III) -, V(IV) -, and V(V) -dipic complexes interact with and penetrate the lipid interfaces differently, an effect that agrees with the compounds' efficacy at lowering elevated blood glucose levels in diabetic rats. PMID:24615733

  12. Phospholipid flippases: building asymmetric membranes and transport vesicles

    PubMed Central

    Sebastian, Tessy T.; Baldridge, Ryan D.; Xu, Peng; Graham, Todd R.

    2012-01-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. PMID:22234261

  13. Cholesterol interactions with tetracosenoic acid phospholipids in model cell membranes: role of the double-bond position.

    PubMed

    Ayanoglu, E; Chiche, B H; Beatty, M; Djerassi, C; Düzgüneş, N

    1990-04-10

    The synthesis and thermotropic properties of 1,2-di-(9Z)-9-tetracosenoylphosphatidylcholine [delta 9-PC(24:1,24:1), 1], 1,2-di-(5Z)-5-tetracosenoylphosphatidylcholine [delta 5-PC(24:1,24:1), 2], and 1,2-di-(15Z)-15- tetracosenoylphosphatidylcholine [delta 15-PC(24:1,24:1), 3] are reported. Liposomes prepared from these phospholipids differ from those of the natural sponge phospholipids, 1,2-di-(5Z,9Z)-5,9-hexacosadienoylphosphatidylcholine (4a) and the corresponding ethanolamine (4b), both of which virtually exclude cholesterol from their bilayers. The behavior of 1 and 2 is similar to that of 1,2-di-(6Z,9Z)-6,9-hexacosadienoylphosphatidylcholine (5), which exhibits a partial molecular interaction with cholesterol. In the case of 3, cholesterol appears to interact with the saturated acyl chain regions of this phospholipid in a manner similar to that of its interaction with DPPC acyl chains. This study delineates the effect of the double-bond location in long fatty acyl chains of phospholipids on their interactions with cholesterol. PMID:2354147

  14. p53 attenuates AKT signaling by modulating membrane phospholipid composition

    PubMed Central

    Rueda-Rincon, Natalia; Bloch, Katarzyna; Derua, Rita; Vyas, Rajesh; Harms, Amy; Hankemeier, Thomas; Khan, Niamat Ali; Dehairs, Jonas; Bagadi, Muralidhararao; Binda, Maria Mercedes; Waelkens, Etienne; Marine, Jean-Christophe; Swinnen, Johannes V.

    2015-01-01

    The p53 tumor suppressor is the central component of a complex network of signaling pathways that protect organisms against the propagation of cells carrying oncogenic mutations. Here we report a previously unrecognized role of p53 in membrane phospholipids composition. By repressing the expression of stearoyl-CoA desaturase 1, SCD, the enzyme that converts saturated to mono-unsaturated fatty acids, p53 causes a shift in the content of phospholipids with mono-unsaturated acyl chains towards more saturated phospholipid species, particularly of the phosphatidylinositol headgroup class. This shift affects levels of phosphatidylinositol phosphates, attenuates the oncogenic AKT pathway, and contributes to the p53-mediated control of cell survival. These findings expand the p53 network to phospholipid metabolism and uncover a new molecular pathway connecting p53 to AKT signaling. PMID:26061814

  15. Phosphocreatine Interacts with Phospholipids, Affects Membrane Properties and Exerts Membrane-Protective Effects

    PubMed Central

    Tokarska-Schlattner, Malgorzata; Epand, Raquel F.; Meiler, Flurina; Zandomeneghi, Giorgia; Neumann, Dietbert; Widmer, Hans R.; Meier, Beat H.; Epand, Richard M.; Saks, Valdur; Wallimann, Theo; Schlattner, Uwe

    2012-01-01

    A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state 31P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could

  16. Interactions of lauryl gallate with phospholipid components of biological membranes.

    PubMed

    Jurak, Małgorzata; Miñones, José

    2016-08-01

    The effect of different amounts of lauryl gallate (LG) on properties of the model membranes of phosphatidylcholines (PC), differing in the presence of double bonds in the hydrocarbon chains, and phosphatidylglycerol (PG) was described in terms of phase behaviour of mixtures, interactions between both components, monolayers stability and their organization. The Langmuir monolayer technique was used to monitor the surface thermodynamics (i.e. the excess area and excess Gibbs energy of mixing) on the basis of surface pressure-area per molecule (π-A) isotherms. Simultaneously, morphology of the studied monolayers was visualized by the Brewster angle microscopy (BAM). This allowed evaluating the kind and magnitude of interactions which influence on the phase behaviour and structural properties of the monolayers. The obtained results can be helpful to reveal the mechanism of phospholipid antioxidant protection and important pharmacological (antimicrobial) role of lauryl gallate for production of effective therapeutic substances. PMID:27117642

  17. Coverage and disruption of phospholipid membranes by oxide nanoparticles.

    PubMed

    Pera, Harke; Nolte, Tom M; Leermakers, Frans A M; Kleijn, J Mieke

    2014-12-01

    We studied the interactions of silica and titanium dioxide nanoparticles with phospholipid membranes and show how electrostatics plays an important role. For this, we systematically varied the charge density of both the membranes by changing their lipid composition and the oxide particles by changing the pH. For the silica nanoparticles, results from our recently presented fluorescence vesicle leakage assay are combined with data on particle adsorption onto supported lipid bilayers obtained by optical reflectometry. Because of the strong tendency of the TiO2 nanoparticles to aggregate, the interaction of these particles with the bilayer was studied only in the leakage assay. Self-consistent field (SCF) modeling was applied to interpret the results on a molecular level. At low charge densities of either the silica nanoparticles or the lipid bilayers, no electrostatic barrier to adsorption exists. However, the adsorption rate and adsorbed amounts drop with increasing (negative) charge densities on particles and membranes because of electric double-layer repulsion, which is confirmed by the effect of the ionic strength. SCF calculations show that charged particles change the structure of lipid bilayers by a reorientation of a fraction of the zwitterionic phosphatidylcholine (PC) headgroups. This explains the affinity of the silica particles for pure PC lipid layers, even at relatively high particle charge densities. Particle adsorption does not always lead to the disruption of the membrane integrity, as is clear from a comparison of the leakage and adsorption data for the silica particles. The attraction should be strong enough, and in line with this, we found that for positively charged TiO2 particles vesicle disruption increases with increasing negative charge density on the membranes. Our results may be extrapolated to a broader range of oxide nanoparticles and ultimately may be used for establishing more accurate nanoparticle toxicity assessments and drug

  18. The effect of hydroxylated fatty acid-containing phospholipids in the remodeling of lipid membranes.

    PubMed

    Piotto, Stefano; Trapani, Alfonso; Bianchino, Erminia; Ibarguren, Maitane; López, David J; Busquets, Xavier; Concilio, Simona

    2014-06-01

    The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. PMID:24463068

  19. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

    Radoicic, Jasmina; Lu, George J.; Opella, Stanley J.

    2014-01-01

    Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function. PMID:25032938

  20. Influence of ibuprofen on phospholipid membranes

    NASA Astrophysics Data System (ADS)

    Jaksch, Sebastian; Lipfert, Frederik; Koutsioubas, Alexandros; Mattauch, Stefan; Holderer, Olaf; Ivanova, Oxana; Frielinghaus, Henrich; Hertrich, Samira; Fischer, Stefan F.; Nickel, Bert

    2015-02-01

    A basic understanding of biological membranes is of paramount importance as these membranes comprise the very building blocks of life itself. Cells depend in their function on a range of properties of the membrane, which are important for the stability and function of the cell, information and nutrient transport, waste disposal, and finally the admission of drugs into the cell and also the deflection of bacteria and viruses. We have investigated the influence of ibuprofen on the structure and dynamics of L-α -phosphatidylcholine (SoyPC) membranes by means of grazing incidence small-angle neutron scattering, neutron reflectometry, and grazing incidence neutron spin echo spectroscopy. From the results of these experiments, we were able to determine that ibuprofen induces a two-step structuring behavior in the SoyPC films, where the structure evolves from the purely lamellar phase for pure SoyPC over a superposition of two hexagonal phases to a purely hexagonal phase at high concentrations. A relaxation, which is visible when no ibuprofen is present in the membrane, vanishes upon addition of ibuprofen. This we attribute to a stiffening of the membrane. This behavior may be instrumental in explaining the toxic behavior of ibuprofen in long-term application.

  1. sup 31 Phosphorus NMR studies of renal membrane phospholipids

    SciTech Connect

    Boylan, J.G.

    1988-01-01

    A research program was undertaken in order to study the effects of toxin-induced acute renal failure and streptozotozin-induced diabetes mellitus on the organization and motion of phospholipids in kidney membranes. The method of study included {sup 31}P NMR and computer simulated lineshape analyses and apparent lipid diffusion rate measurements. These were performed with selected tissue samples and disease models, and the results were compared with those of healthy renal tissues in order to identify changes associated with the diseased state. A new selective-excitation NMR pulse sequence was developed in which a portion of the lipid molecules were excited with a series of short equally-spaced RF pulses. As the position of the molecules changed on the membrane by lateral diffusion, the shape of the hole was seen to change. Experimental spectra were compared with calculated lineshapes in order to obtain estimates of useful molecular parameters including relation times and lateral lipid diffusion rates. The lineshape calculations were based on a solution to the Bloch equations for complex magnetization components with exchange terms added for diffusion.

  2. Proton/hydroxide conductance and permeability through phospholipid bilayer membranes.

    PubMed Central

    Gutknecht, J

    1987-01-01

    Proton/hydroxide (H+/OH-) permeability of phospholipid bilayers is several orders of magnitude higher than alkali or halide ion permeabilities at pH 7. The objective of this study was to determine the mechanism(s) of H+/OH- conductance and permeability through planar phospholipid bilayer membranes. Membranes were formed from decane solutions of bacterial phosphatidylethanolamine, diphytanoyl phosphatidylcholine, or egg phosphatidylcholine plus cholesterol. At pH 7, H+/OH- conductance (GH/OH) ranged from 2 to 6 nS.cm-2, corresponding to H+/OH- "net" permeabilities of (0.4-1.6) X 10(-5) cm.sec-1. GH/OH was inhibited by serum albumin (fatty acid-free), phloretin, and low pH. GH/OH was increased by chlorodecane, long-chain fatty acids, and voltages greater than 80 mV. Water permeability and GH/OH were not correlated. The results suggest that the H+/OH- charge carrier (i) is primarily anionic, (ii) crosses the membrane via nonpolar pathway(s), and (iii) can be removed from the membrane by "washing" with serum albumin. The simplest explanation is that the phospholipids contain weakly acidic contaminants that act as proton carriers at neutral pH. However, at low pH or in the presence of inhibitors, a "background" GH/OH remains that may be due to other mechanisms. PMID:2819878

  3. Membrane-derived phospholipids control synaptic neurotransmission and plasticity.

    PubMed

    García-Morales, Victoria; Montero, Fernando; González-Forero, David; Rodríguez-Bey, Guillermo; Gómez-Pérez, Laura; Medialdea-Wandossell, María Jesús; Domínguez-Vías, Germán; García-Verdugo, José Manuel; Moreno-López, Bernardo

    2015-05-01

    Synaptic communication is a dynamic process that is key to the regulation of neuronal excitability and information processing in the brain. To date, however, the molecular signals controlling synaptic dynamics have been poorly understood. Membrane-derived bioactive phospholipids are potential candidates to control short-term tuning of synaptic signaling, a plastic event essential for information processing at both the cellular and neuronal network levels in the brain. Here, we showed that phospholipids affect excitatory and inhibitory neurotransmission by different degrees, loci, and mechanisms of action. Signaling triggered by lysophosphatidic acid (LPA) evoked rapid and reversible depression of excitatory and inhibitory postsynaptic currents. At excitatory synapses, LPA-induced depression depended on LPA1/Gαi/o-protein/phospholipase C/myosin light chain kinase cascade at the presynaptic site. LPA increased myosin light chain phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. At inhibitory synapses, postsynaptic LPA signaling led to dephosphorylation, and internalization of the GABAAγ2 subunit through the LPA1/Gα12/13-protein/RhoA/Rho kinase/calcineurin pathway. However, LPA-induced depression of GABAergic transmission was correlated with an endocytosis-independent reduction of GABAA receptors, possibly by GABAAγ2 dephosphorylation and subsequent increased lateral diffusion. Furthermore, endogenous LPA signaling, mainly via LPA1, mediated activity-dependent inhibitory depression in a model of experimental synaptic plasticity. Finally, LPA signaling, most likely restraining the excitatory drive incoming to motoneurons, regulated performance of motor output commands, a basic brain processing task. We propose that lysophospholipids serve as potential local messengers that tune synaptic strength to precedent activity of the neuron. PMID:25996636

  4. Force Spectroscopy Reveals the Effect of Different Ions in the Nanomechanical Behavior of Phospholipid Model Membranes: The Case of Potassium Cation

    PubMed Central

    Redondo-Morata, Lorena; Oncins, Gerard; Sanz, Fausto

    2012-01-01

    How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes. PMID:22225799

  5. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  6. Effect of antimicrobial peptides from Australian tree frogs on anionic phospholipid membranes.

    PubMed

    Gehman, John D; Luc, Fiona; Hall, Kristopher; Lee, Tzong-Hsien; Boland, Martin P; Pukala, Tara L; Bowie, John H; Aguilar, Marie-Isabel; Separovic, Frances

    2008-08-19

    Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface

  7. Responding phospholipid membranes--interplay between hydration and permeability.

    PubMed Central

    Sparr, E; Wennerström, H

    2001-01-01

    Osmotic forces are important in regulating a number of physiological membrane processes. The effect of osmotic pressure on lipid phase behavior is of utmost importance for the extracellular lipids in stratum corneum (the outer part of human skin), due to the large gradient in water chemical potential between the water-rich tissue on the inside, and the relative dry environment on the outside of the body. We present a theoretical model for molecular diffusional transport over an oriented stack of two-component lipid bilayers in the presence of a gradient in osmotic pressure. This gradient serves as the driving force for diffusional motion of water. It also causes a gradient in swelling and phase transformations, which profoundly affect the molecular environment and thus the local diffusion properties. This feedback mechanism generates a nonlinear transport behavior, which we illustrate by calculations of the flux of water and solute (nicotine) through the bilayer stack. The calculated water flux shows qualitative agreement with experimental findings for water flux through stratum corneum. We also present a physical basis for the occlusion effect. Phase behavior of binary phospholipid mixtures at varying osmotic pressures is modeled from the known interlamellar forces and the regular solution theory. A first-order phase transformation from a gel to a liquid--crystalline phase can be induced by an increase in the osmotic pressure. In the bilayer stack, a transition can be induced along the gradient. The boundary conditions in water chemical potential can thus act as a switch for the membrane permeability. PMID:11463643

  8. Phospholipid transport via mitochondria

    PubMed Central

    Tamura, Yasushi; Sesaki, Hiromi; Endo, Toshiya

    2014-01-01

    In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. Since cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial outer and inner membranes. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms. PMID:24954234

  9. Mammalian phospholipid homeostasis: evidence that membrane curvature elastic stress drives homeoviscous adaptation in vivo.

    PubMed

    Dymond, Marcus K

    2016-08-01

    Several theories of phospholipid homeostasis have postulated that cells regulate the molecular composition of their bilayer membranes, such that a common biophysical membrane parameter is under homeostatic control. Two commonly cited theories are the intrinsic curvature hypothesis, which states that cells control membrane curvature elastic stress, and the theory of homeoviscous adaptation, which postulates cells control acyl chain packing order (membrane order). In this paper, we present evidence from data-driven modelling studies that these two theories correlate in vivo. We estimate the curvature elastic stress of mammalian cells to be 4-7 × 10(-12) N, a value high enough to suggest that in mammalian cells the preservation of membrane order arises through a mechanism where membrane curvature elastic stress is controlled. These results emerge from analysing the molecular contribution of individual phospholipids to both membrane order and curvature elastic stress in nearly 500 cellular compositionally diverse lipidomes. Our model suggests that the de novo synthesis of lipids is the dominant mechanism by which cells control curvature elastic stress and hence membrane order in vivo These results also suggest that cells can increase membrane curvature elastic stress disproportionately to membrane order by incorporating polyunsaturated fatty acids into lipids. PMID:27534697

  10. Association of the polioviral RNA polymerase complex with phospholipid membranes.

    PubMed Central

    Butterworth, B E; Shimshick, E J; Yin, F H

    1976-01-01

    Polioviral RNA polymerase complex, which consists of enzyme, template, and nascent RNA, is membrane bound in vivo. The solubilized RNA polymerase complex associated spontaneously in vitro with phospholipid bilayer membranes (liposomes) of defined composition. The degree of association at 37 degrees C was greater for those membranes that were more fluid, suggesting that the binding involves the interaction of the RNA polymerase complex with the hydrocarbon chains in the interior of the lipid bilayer. The polymerase activity was not enhanced by addition of the lipid; in fact, the addition of some of the longer-chain lipids resulted in up to a 40% inhibition of the polymerase activity. Spin-label electron paramagnetic resonance experiments, which measured the membrane fluidity, and kinetic experiments on the rate of incorporation of tritiated UTP into RNA by the polymerase were performed as a function of temperature. The results indicated that the activity of the polymerase was not affected by the physical state of the phospholipid membrane and that its active site was not intimately associated with the membrane. Analysis of both the viral and host polypeptides associated with the smooth membrane-bound polymerase indicated that X was the primary viral polypeptide present. In addition, host polypeptides of molecular weight 86,000, 62,000, 54,000, and 46,000 were also present. If the membrane was disrupted with detergent, polypeptide X was released from the polymerase activity, suggesting that X may play a role in binding the polymerase to the membrane. In an analogous manner, polypeptide X associated spontaneously with phospholipid membranes to a greater extent than the capsid polypeptides. Analysis of both the host and viral polypeptides associated with the viral RNA polymerase purified by precipitation in 2 M LiCl indicated that host polypeptides of molecular weight 106,000, 38,000, 33,000, and 14,000 were the major constituents, whereas relatively small amounts of

  11. Extraction of Phospholipids from Human Erythrocyte Membranes by Hemoglobin Oxidation Products.

    PubMed

    Brunauer, Linda S; Chen, James Y; Koontz, M Zachary; Davis, Kathryn K; O'Brien, Laura E; Wright, Emily M; Huestis, Wray H

    2016-06-01

    This investigation examines oxidation conditions under which hemoglobin extracts membrane phospholipid from erythrocytes and model membranes. In erythrocytes made echinocytic with exogenous phospholipid, addition of hemoglobin oxidized with hydrogen peroxide (H2O2) or Vitamin C (conditions that result in the formation of significant quantities of choleglobin), but not ferricyanide (which produces predominantly methemoglobin), induced dose-dependent shape reversion to less echinocytic forms, consistent with extraction of phospholipids from the exofacial side of the membrane. Erythrocytes preloaded with radiolabeled phosphatidylcholine or NBD-labeled phosphatidylcholine, phosphatidylglycerol or phosphatidic acid, exhibited greatest extraction of radiolabel or fluorescence signal with exogenous hemoglobin oxidized via H2O2 or Vitamin C, but not ferricyanide. However, with NBD-phosphatidylserine (a preferential inner monolayer intercalator), significantly less extraction of labeled lipid occurred with oxidized hemoglobin prepared under all three oxidizing conditions. In dimyristoylphosphatidylcholine liposomes containing radiolabeled phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine, subsequent addition of hemoglobin oxidized with H2O2 or Vitamin C extracted radiolabeled lipid with significantly greater efficiency than ferricyanide-treated hemoglobin, with enhanced extraction detectable at levels approaching physiological plasma oxidant concentrations. Radiolabeled lipid extraction was comparable for phospholipids containing saturated acyl chains between 12 and 18 carbons but diminished significantly for oleoyl-containing phospholipids. Hemoglobin dimerization occurred at very low levels with H2O2 treatment, and even lower levels with Vitamin C treatment, and thus did not correlate to the high efficiency and consistent levels of lipid extraction observed with these treatments. These findings indicate that choleglobin extracts lipids from cell

  12. Trans Fatty Acid Derived Phospholipids Show Increased Membrane Cholesterol and Reduced Receptor Activation as Compared to Their Cis Analogs

    PubMed Central

    Niu, Shui-Lin; Mitchell, Drake C.; Litman, Burton J.

    2005-01-01

    The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid–cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid–receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA–phospholipid could be factors contributing to the elevation of LDL cholesterol. PMID:15766276

  13. Interaction of enterocyte FABPs with phospholipid membranes: clues for specific physiological roles.

    PubMed

    Falomir-Lockhart, Lisandro J; Franchini, Gisela R; Guerbi, María Ximena; Storch, Judith; Córsico, Betina

    2011-01-01

    Intestinal and liver fatty acid binding proteins (IFABP and LFABP, respectively) are cytosolic soluble proteins with the capacity to bind and transport hydrophobic ligands between different sub-cellular compartments. Their functions are still not clear but they are supposed to be involved in lipid trafficking and metabolism, cell growth, and regulation of several other processes, like cell differentiation. Here we investigated the interaction of these proteins with different models of phospholipid membrane vesicles in order to achieve further insight into their specificity within the enterocyte. A combination of biophysical and biochemical techniques allowed us to determine affinities of these proteins to membranes, the way phospholipid composition and vesicle size and curvature modulate such interaction, as well as the effect of protein binding on the integrity of the membrane structure. We demonstrate here that, besides their apparently opposite ligand transfer mechanisms, both LFABP and IFABP are able to interact with phospholipid membranes, but the factors that modulate such interactions are different for each protein, further implying different roles for IFABP and LFABP in the intracellular context. These results contribute to the proposed central role of intestinal FABPs in the lipid traffic within enterocytes as well as in the regulation of more complex cellular processes. PMID:21539932

  14. Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids

    PubMed Central

    Pignataro, María Florencia; Dodes-Traian, Martín M.; González-Flecha, F. Luis; Sica, Mauricio; Mangialavori, Irene C.; Rossi, Juan Pablo F. C.

    2015-01-01

    The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca2+ pump (PMCA). We found that Ca2+-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca2+-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA. PMID:25605721

  15. Phospholipid Metabolism and Membrane Synthesis During Sporulation in Bacillus megaterium

    PubMed Central

    Bacon, Karen; White, David

    1974-01-01

    In view of previously published reports of localized membrane growth in exponentially growing Bacillus megaterium and in sporulating Bacillus cereus, an attempt was made to describe phospholipid metabolism and the topology of membrane synthesis during sporulation in B. megaterium. The cells were pulsed with radioactive glycerol or acetate at the time of septum formation, and the specific activity of the lipid fraction was measured at various times through the free spore stage. The bulk of the material labeled during septation could not be recovered in the spore. Rather, it was found that the labeled lipid fraction underwent considerable turnover during spore development. Additionally, other experiments revealed that the lipid made before the initiation of sporulation was also subject to extensive turnover. In order to minimize both the confounding effects of lipid turnover and the possible presence of lateral diffusion of labeled lipid in the membrane, autoradiography of cells pulse labeled with radioactive glycerol at the time of septation was performed; a symmetrical grain distribution resulted. Thus, despite previously published suggestions to the contrary, the current experimental techniques could not demonstrate the existence of localized membrane synthesis in B. megaterium during sporulation. PMID:4206872

  16. Probing Lipid Membrane Rafts (Microdomains) with Fluorescent Phospholipids

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Mitchel, Drake

    2011-10-01

    Membrane rafts are enriched in sphingolipids and cholesterol, they exist in a more ordered state (the liquid-ordered phase; lo) than the bulk membrane (the liquid-disordered phase; ld). Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form membrane rafts over a wide range of molar ratios. We are examining the ability of two fluorescent probes, NBD linked to di-16:0 PE which partitions into the lo phase, and NBD linked to di-18:1 PE which partitions into the ld phase, to detect these two phases. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the size and stability of POPC/SPM/Chol membrane rafts. We report on the fluorescence lifetime and anisotropy decay dynamics of two fluorescent probes. Data were acquired via frequency-domain measurements from 5 to 250 MHz.

  17. Reconstitution of the membrane protein OmpF into biomimetic block copolymer-phospholipid hybrid membranes.

    PubMed

    Bieligmeyer, Matthias; Artukovic, Franjo; Nussberger, Stephan; Hirth, Thomas; Schiestel, Thomas; Müller, Michaela

    2016-01-01

    Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  18. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    PubMed Central

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  19. Fatty acid remodeling by LPCAT3 enriches arachidonate in phospholipid membranes and regulates triglyceride transport

    PubMed Central

    Hashidate-Yoshida, Tomomi; Harayama, Takeshi; Hishikawa, Daisuke; Morimoto, Ryo; Hamano, Fumie; Tokuoka, Suzumi M; Eto, Miki; Tamura-Nakano, Miwa; Yanobu-Takanashi, Rieko; Mukumoto, Yoshiko; Kiyonari, Hiroshi; Okamura, Tadashi; Kita, Yoshihiro; Shindou, Hideo; Shimizu, Takao

    2015-01-01

    Polyunsaturated fatty acids (PUFAs) in phospholipids affect the physical properties of membranes, but it is unclear which biological processes are influenced by their regulation. For example, the functions of membrane arachidonate that are independent of a precursor role for eicosanoid synthesis remain largely unknown. Here, we show that the lack of lysophosphatidylcholine acyltransferase 3 (LPCAT3) leads to drastic reductions in membrane arachidonate levels, and that LPCAT3-deficient mice are neonatally lethal due to an extensive triacylglycerol (TG) accumulation and dysfunction in enterocytes. We found that high levels of PUFAs in membranes enable TGs to locally cluster in high density, and that this clustering promotes efficient TG transfer. We propose a model of local arachidonate enrichment by LPCAT3 to generate a distinct pool of TG in membranes, which is required for normal directionality of TG transfer and lipoprotein assembly in the liver and enterocytes. DOI: http://dx.doi.org/10.7554/eLife.06328.001 PMID:25898003

  20. Fatty acid remodeling by LPCAT3 enriches arachidonate in phospholipid membranes and regulates triglyceride transport.

    PubMed

    Hashidate-Yoshida, Tomomi; Harayama, Takeshi; Hishikawa, Daisuke; Morimoto, Ryo; Hamano, Fumie; Tokuoka, Suzumi M; Eto, Miki; Tamura-Nakano, Miwa; Yanobu-Takanashi, Rieko; Mukumoto, Yoshiko; Kiyonari, Hiroshi; Okamura, Tadashi; Kita, Yoshihiro; Shindou, Hideo; Shimizu, Takao

    2015-01-01

    Polyunsaturated fatty acids (PUFAs) in phospholipids affect the physical properties of membranes, but it is unclear which biological processes are influenced by their regulation. For example, the functions of membrane arachidonate that are independent of a precursor role for eicosanoid synthesis remain largely unknown. Here, we show that the lack of lysophosphatidylcholine acyltransferase 3 (LPCAT3) leads to drastic reductions in membrane arachidonate levels, and that LPCAT3-deficient mice are neonatally lethal due to an extensive triacylglycerol (TG) accumulation and dysfunction in enterocytes. We found that high levels of PUFAs in membranes enable TGs to locally cluster in high density, and that this clustering promotes efficient TG transfer. We propose a model of local arachidonate enrichment by LPCAT3 to generate a distinct pool of TG in membranes, which is required for normal directionality of TG transfer and lipoprotein assembly in the liver and enterocytes. PMID:25898003

  1. Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers

    PubMed Central

    Fleming, Patrick J.; Freites, J. Alfredo; Moon, C. Preston; Tobias, Douglas J.; Fleming, Karen G.

    2011-01-01

    Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host-guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210

  2. Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas.

    PubMed

    Daubenspeck, James M; Liu, Runhua; Dybvig, Kevin

    2016-01-01

    Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria. PMID:27603308

  3. Phospholipid Membrane Protection by Sugar Molecules during Dehydration-Insights into Molecular Mechanisms Using Scattering Techniques

    SciTech Connect

    Garvey, Christopher J.; Lenné, Thomas; Koster, Karen L.; Kent, Ben; Bryant, Gary

    2014-09-24

    Scattering techniques have played a key role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to study how different molecules (e.g., cholesterol) can affect phospholipid membrane structure. However, there has been much less attention paid to the effects of molecules that remain in the aqueous phase. One important example is the role played by small solutes, particularly sugars, in protecting phospholipid membranes during drying or slow freezing. In this paper, we present new results and a general methodology, which illustrate how contrast variation small angle neutron scattering (SANS) and synchrotron-based X-ray scattering (small angle (SAXS) and wide angle (WAXS)) can be used to quantitatively understand the interactions between solutes and phospholipids. Specifically, we show the assignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the particular example of hexagonal II phases formed by phospholipids.

  4. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    PubMed

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. PMID:27433988

  5. On the formation of a macroscopically flat phospholipid membrane on a hydrosol substrate

    NASA Astrophysics Data System (ADS)

    Tikhonov, A. M.; Asadchikov, V. E.; Volkov, Yu. O.

    2015-10-01

    The dependence of the structure of a phospholipid layer (DSPC and SOPC) adsorbed on a hydrosol substrate on the concentration of NaOH in a solution of 5-nm silica particles has been studied by X-ray reflectrometry with the use of synchrotron radiation. Profiles of the electron density (polarizability) have been reconstructed from the experimental data within a model-independent approach. According to these profiles, the thickness of the lipid film can vary from a monolayer (~35 Å) to several bilayers (~450 Å). At the volume concentration of NaOH of ~0.5 mol/L, the film on the hydrosol surface is a macroscopically flat phospholipid membrane (bilayer) with a thickness of ~60 Å and with areas of (45 ± 2) and (49 ± 3) Å2 per DSPC and SOPC molecule, respectively.

  6. Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop.

    PubMed

    Nakao, Hiroyuki; Ikeda, Keisuke; Ishihama, Yasushi; Nakano, Minoru

    2016-06-21

    The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionite-quenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER. PMID:27332127

  7. Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs*

    PubMed Central

    Morgan, Christopher R.; Hebling, Christine M.; Rand, Kasper D.; Stafford, Darrel W.; Jorgenson, James W.; Engen, John R.

    2011-01-01

    Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. PMID:21715319

  8. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

  9. Interaction of Lipopolysaccharide and Phospholipid in Mixed Membranes: Solid-State 31P-NMR Spectroscopic and Microscopic Investigations

    PubMed Central

    Nomura, Kaoru; Inaba, Takehiko; Morigaki, Kenichi; Brandenburg, Klaus; Seydel, Ulrich; Kusumoto, Shoichi

    2008-01-01

    Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state 31P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-α-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1 M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical

  10. Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids.

    PubMed

    Ramos, R Godoy; Libong, D; Rakotomanga, M; Gaudin, K; Loiseau, P M; Chaminade, P

    2008-10-31

    The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids. PMID:18823632

  11. Forty five years with membrane phospholipids, phospholipases and lipid mediators: A historical perspective.

    PubMed

    Chap, Hugues

    2016-06-01

    Phospholipases play a key role in the metabolism of phospholipids and in cell signaling. They are also a very useful tool to explore phospholipid structure and metabolism as well as membrane organization. They are at the center of this review, covering a period starting in 1971 and focused on a number of subjects in which my colleagues and I have been involved. Those include determination of phospholipid asymmetry in the blood platelet membrane, biosynthesis of lysophosphatidic acid, biochemistry of platelet-activating factor, first attempts to define the role of phosphoinositides in cell signaling, and identification of novel digestive (phospho)lipases such as pancreatic lipase-related protein 2 (PLRP2) or phospholipase B. Besides recalling some of our contributions to those various fields, this review makes an appraisal of the impressive and often unexpected evolution of those various aspects of membrane phospholipids and lipid mediators. It is also the occasion to propose some new working hypotheses. PMID:27059515

  12. [sup 31]P NMR study of immobilized artificial membrane surfaces. Structure and dynamics of immobilized phospholipids

    SciTech Connect

    Qiu, X.; Pidgeon, C. )

    1993-11-25

    Chromatography surfaces were prepared by immobilizing a single-chain ether phospholipid at approximately a monolayer density on silica particles. The chromatography particles are denoted as [sup ether]IAM.PC[sup C10/C3], and they are stable to all solvents. The structure and dynamics of the interphase created by immobilizing phospholipids on silica particles were studied by [sup 31]P NMR methods. [sup ether]IAM.PC[sup C10/C3] spontaneously wets when suspended in both aqueous and organic solvents, and [sup 31]P NMR spectra were obtained in water, methanol, chloroform, acetonitrile, and acetone. [sup 31]P NMR spectra were subjected to line-shape analysis. From line-shape analysis, the correlation times for rapid internal motion ([tau]-PLL) and wobbling ([tau]-PRP) of the phospholipid headgroup were calculated for each solvent. Immobilized phospholipid headgroups comprising the IAM interfacial region undergo rapid reorientation similar to the case of the phospholipids forming liposome membranes with [tau]-PLL approximately 1 ns. Phospholipids in liposome membranes exhibit slower wobbling motion ([tau]-PRP approximately 1 ms) in the plane of the membrane. However, the immobilized phospholipids on [sup ether]IAM.PC[sup C10/C3] surfaces wobble with correlation times [tau]-PRP that depend on the solvent bathing the [sup ether]IAM.PC[sup C10/C3] surface. 41 refs., 9 figs., 2 tabs.

  13. Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy

    PubMed Central

    Tawa, Keiko; Morigaki, Kenichi

    2005-01-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates. PMID:16040759

  14. Nonspecular neutron scattering from highly aligned phospholipid membranes

    NASA Astrophysics Data System (ADS)

    Münster, C.; Salditt, T.; Vogel, M.; Siebrecht, R.; Peisl, J.

    1999-05-01

    We report a neutron scattering study of multilamellar membranes supported on solid substrates. In contrast to previous work, the high degree of orientational alignment allows for a clear distinction between specular and nonspecular reflectivity contributions. In particular, we demonstrate that by using the specific advantages of neutron optics the nonspecular scattering can be mapped over a wide range of reciprocal space. Several orders of magnitude in scattering signal and parallel momentum transfer can easily be recorded in multilamellar stacks of lipid membranes. This opens up the possibility to study fluctuations, and more generally lateral structure parameters of membrane on length scales between a few Å up to several μm. The first results obtained for a system of partially hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) indicate strong deviations from the predictions of the standard Caillé model.

  15. In situ imaging of micropatterned phospholipid membranes by surface plasmon fluorescence microscopy.

    PubMed

    Tawa, Keiko; Morigaki, Kenichi

    2010-12-01

    A parallel microscopic imaging technique, surface plasmon microscopy (SPM)-surface plasmon fluorescence microscopy (SPFM), is introduced as a versatile analytical tool to monitor biochips. In spite of the fact that the fluorescence excited by surface plasmon is 1-2 order stronger compared with the total internal reflection fluorescence microscopy, SPFM has not fully utilized its advantages because fluorescence from fluorophores near the gold surface is almost entirely quenched due to the Förster energy transfer. In this study, SiO2 layer sputtered on the gold substrate suppressed the quenching of fluorescence and enabled a parallel measurement of SPM and SPFM. As a model system, micropatterned membranes composed of polymeric and fluid phospholipid bilayers were employed. The difference of film thickness could be detected by SPM, and SPFM provided information on the composition and structure of membranes, enabling the distinction between polymeric and fluid phospholipid bilayers. These results suggest the general applicability of SPM-SPFM for various formats of biochips. PMID:20728323

  16. The local anesthetic tetracaine destabilizes membrane structure by interaction with polar headgroups of phospholipids.

    PubMed

    Shimooka, T; Shibata, A; Terada, H

    1992-03-01

    The effect of the local anesthetic tetracaine at less than 10 mM on the water permeability of the phospholipid membrane was examined using liposomes composed of various molar ratios of negatively charged cardiolipin to electrically neutral phosphatidylcholine by monitoring their osmotic shrinkage in hypertonic glucose solution at 30 degrees C. The concentration of tetracaine causing the maximum velocity of shrinkage of liposomes increased with increase in the molar ratio of cardiolipin. Tetracaine increased the zeta-potential of the negatively charged liposomal membrane toward the positive side due to the binding of its cationic form to the negatively charged polar headgroups in the membrane. The maximum velocity of water permeation induced by osmotic shock was observed at essentially the same tetracaine concentration giving a zeta-potential of the liposomal membrane of 0 mV. These concentrations were not affected by change in the sort of acyl-chain of phospholipids in the liposomes when their negative charges were the same. These results suggests that the membrane integrity is governed mainly by the electrical charge of phospholipid polar headgroups when phospholipid bilayers are in the highly fluid state, and that positively charged tetracaine molecules neutralize the negative surface charge, lowering the barrier for water permeation through phospholipid bilayers. PMID:1547263

  17. A phospholipid uptake system in the model plant Arabidopsis thaliana.

    PubMed

    Poulsen, Lisbeth R; López-Marqués, Rosa L; Pedas, Pai R; McDowell, Stephen C; Brown, Elizabeth; Kunze, Reinhard; Harper, Jeffrey F; Pomorski, Thomas G; Palmgren, Michael

    2015-01-01

    Plants use solar energy to produce lipids directly from inorganic elements and are not thought to require molecular systems for lipid uptake from the environment. Here we show that Arabidopsis thaliana Aminophospholipid ATPase10 (ALA10) is a P4-type ATPase flippase that internalizes exogenous phospholipids across the plasma membrane, after which they are rapidly metabolized. ALA10 expression and phospholipid uptake are high in the epidermal cells of the root tip and in guard cells, the latter of which regulate the size of stomatal apertures to modulate gas exchange. ALA10-knockout mutants exhibit reduced phospholipid uptake at the root tips and guard cells and are affected in growth and transpiration. The presence of a phospholipid uptake system in plants is surprising. Our results suggest that one possible physiological role of this system is to internalize lysophosphatidylcholine, a signalling lipid involved in root development and stomatal control. PMID:26212235

  18. Growth of giant membrane lobes mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces.

    PubMed

    Suzuki, Kenji; Masuhara, Hiroshi

    2005-01-18

    We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces and a strategy to control the two-dimensional structure of the membrane lobes on a solid surface. The growth of giant membrane lobes was observed on a single-lipid bilayer which spread from a lump of phospholipid deposited on a silica-glass substrate or an oxidized silicon wafer in aqueous solutions of NaCl, KCl, MgCl2, or CaCl2 at relatively high salt concentrations. Most of the membrane lobes were very flat unilamellar tubes elongating from the lump of phospholipid, and their length reached 1 mm in 5 h. Experimental findings clearly indicate that the membrane lobes are adherent to the surface of the single-lipid bilayer and are mechanically elongated from the lump of phospholipid by the sliding motion of the single-lipid bilayer. We could control the two-dimensional structure of the membrane lobes on the substrate by controlling the spreading direction of the single-lipid bilayer using Pt micropatterns that were deposited on the smooth surface of the oxidized silicon wafer. PMID:15641821

  19. Aspirin, acetaminophen and proton transport through phospholipid bilayers and mitochondrial membranes.

    PubMed

    Gutknecht, J

    1992-09-01

    Mechanisms of proton transport were investigated in planar phospholipid bilayer membranes exposed to aspirin (acetylsalicylic acid), acetaminophen (4-acetamidophenol), benzoic acid and three aspirin metabolites (salicylic acid, gentisic acid and salicyluric acid). The objectives were to characterize the conductances and permeabilities of these weak acids in lipid bilayer membranes and then predict their effects on mitochondrial membranes. Of the compounds tested only aspirin, benzoate and salicylate caused significant increases in membrane conductance. The conductance was due mainly to proton current at low pH and to weak acid anion current at neutral pH. Analysis of the concentration and pH dependence suggests that these weak acids act as HA-2-type proton carriers when pH approximately pK and as lipid soluble anions at neutral pH. Salicylate is much more potent than aspirin and benzoate because salicylate contains an internal hydrogen bond which delocalizes the negative charge and increases the permeability of the anion. Model calculations for mitochondria suggest that salicylate causes net H+ uptake by a cyclic process of HA influx and A- efflux. This model can explain the salicylate-induced uncoupling and swelling observed in isolated mitochondria. Since ingested aspirin breaks down rapidly to form salicylate, these results may clarify the mechanisms of aspirin toxicity in humans. The results may also help to explain why the ingestion of aspirin but not acetaminophen is associated with Reye's syndrome, a disease characterized by impaired energy metabolism and mitochondrial swelling. PMID:1334228

  20. Dissipative Particle Dynamics Simulations for Phospholipid Membranes Based on a Four-To-One Coarse-Grained Mapping Scheme

    PubMed Central

    Li, Xiaoxu; Gao, Lianghui; Fang, Weihai

    2016-01-01

    In this article, a new set of parameters compatible with the dissipative particle dynamics (DPD) force field is developed for phospholipids. The coarse-grained (CG) models of these molecules are constructed by mapping four heavy atoms and their attached hydrogen atoms to one bead. The beads are divided into types distinguished by charge type, polarizability, and hydrogen-bonding capacity. First, we derive the relationship between the DPD repulsive force and Flory-Huggins χ-parameters based on this four-to-one CG mapping scheme. Then, we optimize the DPD force parameters for phospholipids. The feasibility of this model is demonstrated by simulating the structural and thermodynamic properties of lipid bilayer membranes, including the membrane thickness, the area per lipid, the lipid tail orientation, the bending rigidity, the rupture behavior, and the potential of mean force for lipid flip-flop. PMID:27137463

  1. Dissipative Particle Dynamics Simulations for Phospholipid Membranes Based on a Four-To-One Coarse-Grained Mapping Scheme.

    PubMed

    Li, Xiaoxu; Gao, Lianghui; Fang, Weihai

    2016-01-01

    In this article, a new set of parameters compatible with the dissipative particle dynamics (DPD) force field is developed for phospholipids. The coarse-grained (CG) models of these molecules are constructed by mapping four heavy atoms and their attached hydrogen atoms to one bead. The beads are divided into types distinguished by charge type, polarizability, and hydrogen-bonding capacity. First, we derive the relationship between the DPD repulsive force and Flory-Huggins χ-parameters based on this four-to-one CG mapping scheme. Then, we optimize the DPD force parameters for phospholipids. The feasibility of this model is demonstrated by simulating the structural and thermodynamic properties of lipid bilayer membranes, including the membrane thickness, the area per lipid, the lipid tail orientation, the bending rigidity, the rupture behavior, and the potential of mean force for lipid flip-flop. PMID:27137463

  2. Self-quenching of nitrobenzoxadiazole labeled phospholipids in lipid membranes

    NASA Astrophysics Data System (ADS)

    Brown, R. Stephen; Brennan, John D.; Krull, Ulrich J.

    1994-04-01

    The emission intensity, wavelength, and lifetime of the fluorophore nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) are sensitive to the local environmental structure when this species is present as a component of an amphiphilic membrane. Alterations of the physical and electrostatic structure of a membrane can result in changes in the fluorescence signal owing to changes in the extent of self-quenching of the probe. To investigate self-quenching, NBD-PE was incorporated into monolayers and vesicles composed of Egg phosphatidylcholine at concentrations of 0.1 to 50 mol %. Monolayer samples were dipcast onto glass slides at a pressure of 35 mN m-1. Both the integrated intensity per fluorophore (quantum yield) from vesicles and dipcast monolayers, and the mean fluorescence lifetime from vesicles decreased as the concentration of fluorophore in the membranes was increased. At all concentrations studied the decay of NBD-PE fluorescence was fitted to two discrete exponentials, and both lifetime components were observed to change with concentration. The complexity of the fluorescence decay did not permit the use of standard theoretical models such as the Klafter-Blumen or Stern-Volmer equations which are normally employed to describe changes in fluorescence lifetime with changes in quencher concentration. Instead, a phenomenological approach was used to develop an empirical model of fluorescence self-quenching which could describe the observed alterations in the fluorescence lifetime and intensity. The model was based on a combination of Perrin quenching and Förster energy transfer. The fluorescence data was fit by a model wherein NBD-PE formed nonemissive trap sites with a critical radius of Rc=1.0±0.1 nm (Perrin quenching), with Förster energy transfer occurring to the trap sites with an R0 value of 2.55±0.10 nm as determined from spectral overlap integrals.

  3. The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the phospholipid headgroup electrometer concept to phosphatidylserine

    SciTech Connect

    de Kroon, A.I.P.M.; Killian, J.A.; de Gier, J.; de Kruijff, B. )

    1991-01-29

    Deuterium nuclear magnetic resonance ({sup 2}H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the {beta}-position of the serine moiety ((2-{sup 2}H)DOPS) or at the 11-position of the acyl chains ((11,11-{sup 2}H{sub 2})DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH and H-Arg-Met-Leu-Trp-Ala-OH and contain a positive charge of +1 or +2 at the amino terminus or one positive charge at each end of the molecule. Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilyers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting of both (2-{sup 2}H)DOPS and (11,11-{sup 2}H{sub 2})DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. Titrations of DOPS with poly(L-lysine){sub 100}, which were included for reasons of comparison, reveal increased {Delta}v{sub q} values. When the peptide-lipid titrations are carried out without applying a freeze-thaw procedure to achieve full equilibration, two-component {sup 2}H NMR spectra occur. The apparently limited accessibility of the lipid to the peptides under these circumstances is discussed in relation to the ability of the peptides to exhibit transbilayer movement. {sup 2}H spin-lattice relaxation time T1 measurements demonstrate a decrease of the rates of motion of both headgroup and acyl chains of DOPS in the presence of the peptides.

  4. Molecular simulations of the effects of phospholipid and cholesterol peroxidation on lipid membrane properties.

    PubMed

    Neto, Antenor J P; Cordeiro, Rodrigo M

    2016-09-01

    Non-enzymatic lipid peroxidation may change biomembrane structure and function. Here, we employed molecular dynamics simulations to study the effects of either phospholipid or cholesterol peroxidation individually, as well as the combined peroxidation of both components. When lipids were peroxidized, the generated OOH groups migrated to the membrane surface and engaged in H-bonds with each other and the phospholipid carbonyl ester groups. It caused the sn-2 acyl chains of phospholipid hydroperoxides to bend and the whole sterol backbone of cholesterol hydroperoxides to tilt. When phospholipids were kept intact, peroxidation of the sterol backbone led to a partial degradation of its condensing and ordering properties, independently of the position and isomerism of the OOH substitution. However, even in massively peroxidized membranes in which all phospholipids and cholesterol were peroxidized, the condensing and ordering properties of the sterol backbone were still significant. The possible implications for the formation of membrane lateral domains were discussed. Cholesterol peroxyl radicals were also investigated and we found that the OO groups did not migrate to the headgroups region. PMID:27349733

  5. Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.

    PubMed

    Ciaffoni, Fiorella; Tatti, Massimo; Salvioli, Rosa; Vaccaro, Anna Maria

    2003-08-01

    Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D-particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as G(M1). At appropriate Sap D concentrations Cer and G(M1) are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids. PMID:12733985

  6. The Effect of Trans Unsaturation on Molecular Organization in a Phospholipid Membrane

    NASA Astrophysics Data System (ADS)

    Soni, Smita; Runyan, Jenniffer; Brich, Garrison; Ward, Jesse; Sen, Stephanie; Feller, Scott; Wassall, Stephen

    2009-05-01

    The ingestion of trans fatty acids (TFA) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. We synthesized deuterated analogs of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC) that contains a single ``unnatural'' trans double bond and 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC) that contains a single ``natural'' cis double bond. Solid state ^2H NMR spectra for model membranes prepared from these phospholipids reveal a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. In the liquid crystalline, however, the difference between the trans and cis isomers is subtle. Order as probed by the perdeuterated [^2H31]18:0 sn-2 chain, and corroborated by molecular dynamics (MD) simulation, coincides within <5%.

  7. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

    PubMed Central

    Parasassi, T; De Stasio, G; d'Ubaldo, A; Gratton, E

    1990-01-01

    The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns. PMID:2393703

  8. Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

    PubMed

    Kuznetsov, A S; Dubovskii, P V; Vorontsova, O V; Feofanov, A V; Efremov, R G

    2014-05-01

    Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin

  9. Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

    PubMed Central

    Tata, J. R.

    1970-01-01

    1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [32P]orthophosphate, [14C]choline and [14C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with 32P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with 32P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth

  10. Supported Phospholipid Membrane Formation as a Function of 1,2-Dimyristoylphosphatidylglycerol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability to form a reasonably supported phospholipid membrane (SPB) on a surface is vital to our development of a bioreactor that entails electron transfer proteins immobilized on a thiol-modified surface. In this respect, we will present work where we investigated what the influence of calcium ...

  11. Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

    PubMed Central

    Kleinschmidt, J H; Marsh, D

    1997-01-01

    The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide. PMID:9370448

  12. Dynamic membrane protein topological switching upon changes in phospholipid environment

    PubMed Central

    Vitrac, Heidi; MacLean, David M.; Jayaraman, Vasanthi; Bogdanov, Mikhail; Dowhan, William

    2015-01-01

    A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids. PMID:26512118

  13. Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.

    PubMed

    Cohen, F S; Akabas, M H; Zimmerberg, J; Finkelstein, A

    1984-03-01

    It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment. PMID:6699081

  14. Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups*

    PubMed Central

    Yuan, Quan; Kaylor, Joanna J.; Miu, Anh; Bassilian, Sara; Whitelegge, Julian P.; Travis, Gabriel H.

    2010-01-01

    Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant. PMID:19892706

  15. Dynamics of bio-membranes investigated by neutron spin echo: Effects of phospholipid conformations and presence of lidocaine

    NASA Astrophysics Data System (ADS)

    Yi, Zheng

    Bio-membranes of the natural living cells are made of bilayers of phospholipids molecules embedded with other constituents, such as cholesterol and membrane proteins, which help to accomplish a broad range of functions. Vesicles made of lipid bilayers can serve as good model systems for bio-membranes. Therefore these systems have been extensively characterized and much is known about their shape, size, porosity and functionality. In this dissertation we report the studies of the effects of the phosoholipid conformation, such as hydrocarbon number and presence of double bond in hydrophobic tails on dynamics of phospholipids bilayers studied by neutron spin echo (NSE) technique. We have investigated how lidocaine, the most medically used local anesthetics (LA), influence the structural and dynamical properties of model bio-membranes by small angle neutron scattering (SANS), NSE and differential scanning calorimetry (DSC). To investigate the influence of phospholipid conformation on bio-membranes, the bending elasticities kappac of seven saturated and monounsaturated phospholipid bilayers were investigated by NSE spectroscopy. kappa c of phosphatidylcholines (PCS) in liquid crystalline (L alpha) phase ranges from 0.38x10-19 J for 1,2-Dimyristoyl- sn-Glycero-3-Phosphocholine (14:0 PC) to 0.64x10-19 J for 1,2-Dieicosenoyl-sn-Glycero-3-Phosphocholine (20:1 PC). It was confirmed that when the area modulus KA varies little with chain unsaturation or length, the elastic ratios (kappac/ KA)1/2 of bilayers varies linearly with lipid hydrophobic thickness d. For the study of the influence of LA on bio-membranes, SANS measurements have been performed on 14:0 PC bilayers with different concentrations of lidocaine to determine the bilayer thickness dL as a function of the lidocaine concentration. NSE has been used to study the influence of lidocaine on the bending elasticity of 14:0 PC bilayers in Lalpha and ripple gel (Pbeta') phases. Our results confirmed that the molecules of

  16. [Phospholipids and structural modification of tissues and cell membranes for adaptation in high altitude mountains].

    PubMed

    Iakovlev, V M; Vishnevskiĭ, A A; Shanazarov, A S

    2012-01-01

    The nature of the impact of physical factors of high altitudes (3200 m) on the lipids of tissues and membranes of animals was researched. It was established that the adaptation process in Wistar rats was followed by peroxide degradation and subsequent modification of the phospholipids' structure of tissues and microsomal membranes. Adaptive phospholipids reconstruction takes place in microsomal membranes in the tissues of the lungs, brain, liver and skeletal muscles. Together with this, the amount of phosphatidylinositol and phosphatidic acid accumulates, indicating that the hydrolysis of phosphatidylinositol-4, 5 biphosphate to diacylglycerol and secondary messenger--inositol triphosphate, occurs. A decrease in temperature adaptation (+10 degrees C) leads to a more noticeable shift in peroxide oxidation of lipids, phospholipid structure in the tissues and membranes rather than adaptation in thermoneutral conditions (+30 degrees C). Modification of lipid composition of tissues and cell membranes in the highlands obviously increases the adaptive capabilities of cells of the whole body: physical performance and resistance to hypoxia increases in animals. PMID:22586936

  17. Cyclopropyl Sterol and Phospholipid Composition of Membrane Fractions from Maize Roots Treated with Fenpropimorph

    PubMed Central

    Grandmougin, Anne; Bouvier-Navé, Pierrette; Ullmann, Pascaline; Benveniste, Pierre; Hartmann, Marie-Andrée

    1989-01-01

    Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ5-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ5-sterols. PMID:16666813

  18. 25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

    SciTech Connect

    Olsen, Brett N.; Schlesinger, Paul H.; Ory, Daniel S.; Baker, Nathan A.

    2011-02-01

    Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work, using molecular dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers to examine interactions between 25-HC and cholesterol in the same bilayer. When added to cholesterol-containing membranes, 25-HC causes larger changes in membrane structure than when added to cholesterol-free membranes, demonstrating interactions between the two sterols. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and therefore its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These interactions provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through direct modulation of cholesterol availability.

  19. Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli.

    PubMed

    Lin, Yibin; Bogdanov, Mikhail; Tong, Shuilong; Guan, Ziqiang; Zheng, Lei

    2016-01-29

    Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane. PMID:26613781

  20. Binding of monovalent alkali metal ions with negatively charged phospholipid membranes.

    PubMed

    Maity, Pabitra; Saha, Baishakhi; Kumar, Gopinatha Suresh; Karmakar, Sanat

    2016-04-01

    We have systematically investigated the effect of various alkali metal ions with negatively charged phospholipid membranes. Size distributions of large unilamellar vesicles have been confirmed using dynamic light scattering. Zeta potential and effective charges per vesicle in the presence of various alkali metal ions have been estimated from the measured electrophoretic mobility. We have determined the intrinsic binding constant from the zeta potential using electrostatic double layer theory. The reasonable and consistent value of the intrinsic binding constant of Na(+), found at moderate NaCl concentration (10-100 mM), indicates that the Gouy-Chapman theory cannot be applied for very high (> 100mM) and very low (< 10 mM) electrolyte concentrations. The isothermal titration calorimetry study has revealed that the net binding heat of interaction of the negatively charged vesicles with monovalent alkali metal ions is small and comparable to those obtained from neutral phosphatidylcholine vesicles. The overall endothermic response of binding heat suggests that interaction is primarily entropy driven. The entropy gain might arise due to the release of water molecules from the hydration layer vicinity of the membranes. Therefore, the partition model which does not include the electrostatic contribution suffices to describe the interaction. The binding constant of Na(+) (2.4 ± 0.1 M(-1)), obtained from the ITC, is in agreement with that estimated from the zeta potential (-2.0 M(-1)) at moderate salt concentrations. Our results suggest that hydration dynamics may play a vital role in the membrane solution interface which strongly affects the ion-membrane interaction. PMID:26802251

  1. [Relevance of metabolism of membrane phospholipids for Alzheimer dementia].

    PubMed

    Gattaz, W F; Levy, R; Cairns, N J; Förstl, H; Braus, D F; Maras, A

    1996-01-01

    We found a decreased activity of the enzyme phospholipase A2 (PLA2) in brain tissue from 23 patients with Alzheimer's disease (AD) compared to 20 non-demented controls. The decrement was more pronounced in patients with an early onset of disease and correlated significantly with higher counts of senile plaques and neurofibrillary tangles. Decreased PLA2 activity may inhibit the secretion of the amyloid precursor protein (APP) and thus contribute to the formation of the beta-amyloid peptide, the major component of the amyloid plaque in AD. Because PLA2 activity is under genetic control, it is conceivable that the enzyme activity in the brain is related to the activity in blood cells. To test this assumption we investigated PLA2 in platelet membranes from AD patients compared to healthy and psychiatric controls. Platelets are interesting peripheral models in AD research, because they contain and secrete APP. We determined the platelet PLA2 activity in 16 patients with a "probable" AD (NINCDS-ADRDA criteria) as compared to 13 healthy controls and to 14 psychiatric patients with a major depression. There were no significant differences between the three groups regarding age and sex distribution. In the AD patients the cognitive performance was assessed with the CAMCOG and the Mini Mental State Exam (MMSE). The radioenzymatic assay for the determination of PLA2 activity is described elsewhere. Platelet PLA2 activity was significantly reduced in AD patients as compared to healthy (p < 0.03) and to psychiatric controls (p < 0.002). The reduction of the enzyme activity correlated with an early onset of the disease (rs = .43, p < 0.10) and with the cognitive impairment in the CAMCOG (rs = .55, p < 0.05). AD patients with a MMSE-score lower than 10 (median) showed significantly lower PLA2 activity (11.8 +/- 3.1) than patients with MMSE-score higher than 10 (16.2 +/- 4.6, p < 0.05). These findings in platelets are in line with our previous results in brain tissue. In both

  2. [Phospholipid composition and content of the erythrocyte membrane in carriers of sickle cell trait].

    PubMed

    Sarr, N G; Sall, N D; Toure, M; Diatta, A; Seck, I

    1998-01-01

    By its frequency, but also by its morbidity and mortality, sickle cell disease is a real problem of public health in Senegal. It is an inherited disease of the red blood cell, characterized by vaso-occlusive manifestations in several organs, and chronic hemolytic anemia. Out of this work carried upon subjects presenting sickle cell trait (AS), we especially studied the membrane phospholipids. The aims of this study was to identify and to measure the major phospholipids of the red blood cell membrane (lecithin, lysolecithin, cephalin, lysocephalin and sphingomyelin). We noted a significant decrease of the lecithine rates, associated with significant accumulation of lysophosphatidyl choline implied in hemolysis disorders. We have also noted a significant increase of sphingomyelin which might be involved in the adhesive phenomena of the sickle cells. These preliminary results, allow to consider eventual new therapeutics, at least to reduce the clinical disorders of this painful disease. PMID:9827146

  3. Origin of membrane dipole potential: contribution of the phospholipid fatty acid chains.

    PubMed

    Peterson, Uwe; Mannock, David A; Lewis, Ruthven N A H; Pohl, Peter; McElhaney, Ronald N; Pohl, Elena E

    2002-08-01

    The large intrinsic membrane dipole potential, phi(d), is important for protein insertion and functioning as well as for ion transport across natural and model membranes. However, the origin of phi(d) is controversial. From experiments carried out with lipid monolayers, a significant dependence on the fatty acid chain length is suggested, whereas in experiments with lipid bilayers, the contribution of additional -CH(2)-groups seems negligibly small compared with that of the phospholipid carbonyl groups and lipid-bound water molecules. To compare the impact of the -CH(2)-groups of dipalmitoylphosphatidylcholine (DPPC) near and far from the glycerol backbone, we have varied the structure of DPPC by incorporation of sulfur atoms in place of methylene groups in different positions of the fatty acid chain. The phi(d) of symmetric lipid bilayers containing one heteroatom was obtained from the charge relaxation of oppositely charged hydrophobic ions. We have found that the substitution for a S-atom of a -CH(2)-group decreases phi(d). The effect (deltaphi(d) = -22.6 mV) is most pronounced for S-atoms near the lipid head group while a S-atom substitution in the C(13)- or C(14)-position of the hydrocarbon chain does not effect the bilayer dipole potential. Most probably deltaphi(d) does not originate from an altered dipole potential of the acyl chain containing an heteroatom but is mediated by the disruption of chain packing, leading to a decreased density of lipid dipoles in the membrane. PMID:12191841

  4. An Unrecognized Function of Cholesterol: Regulating the Mechanism Controlling Membrane Phospholipid Asymmetry.

    PubMed

    Arashiki, Nobuto; Saito, Masaki; Koshino, Ichiro; Kamata, Kotoe; Hale, John; Mohandas, Narla; Manno, Sumie; Takakuwa, Yuichi

    2016-06-28

    An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling. PMID:27267274

  5. An intercalation mechanism as a mode of action exerted by psychotropic drugs: results of altered phospholipid substrate availabilities in membranes?

    PubMed Central

    Lund, Anders; Pryme, Ian F.; Holmsen, Holm

    2010-01-01

    Patients respond differently to psychotropic drugs, and this is currently a controversial theme among psychiatrists. The effects of 16 psychotropics on cell membrane parameters have been reported. These drugs belong to three major groups used in therapeutic psychiatry: antipsychotics, antidepressants, and anxiolytic/hypnotics. Human platelets, lacking dopamine (D2) receptors (proposed targets of most psychotropics), have been used as a cell model. Here we discuss the effects of these drugs on three metabolic phenomena and also results from Langmuir experiments. Diazepam, in contrast to the remaining drugs, had negligible effects on metabolic phenomena and had no effects in Langmuir experiments. Psychotropic drugs may work through intercalation in membrane phospholipids. It is possible that the fluidity of membranes, rich in essential fatty acids, the content being influenced by diet, could be a contributing factor to the action of psychotropics. This might in turn explain the observed major differences in therapeutic response among patients. PMID:21270935

  6. Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

    PubMed Central

    Bittencourt-Cunha, Paula Rêgo; Silva-Cardoso, Livia; de Oliveira, Giselle Almeida; da Silva, José Roberto; da Silveira, Alan Barbosa; Kluck, George Eduardo Gabriel; Souza-Lima, Michele; Gondim, Katia Calp; Dansa-Petretsky, Marilvia; Silva, Carlos Peres; Masuda, Hatisaburo; da Silva, Mario Alberto Cardoso; Atella, Georgia Correa

    2013-01-01

    In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs. PMID:23827998

  7. Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.

    PubMed Central

    Artmann, G M; Sung, K L; Horn, T; Whittemore, D; Norwich, G; Chien, S

    1997-01-01

    When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time. Images FIGURE 1 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 PMID:9138589

  8. Fibrillation of β amyloid peptides in the presence of phospholipid bilayers and the consequent membrane disruption.

    PubMed

    Qiang, Wei; Yau, Wai-Ming; Schulte, Jürgen

    2015-01-01

    Fibrillation of β amyloid (Aβ) peptides and the accumulation of amyloid plaques are considered as an important clinical hallmark to identify Alzheimer's disease (AD). The physiological connection between Aβ plaques and the disruption of neuronal cells has not been clearly understood. One hypothesis to explain the Aβ neurotoxicity is that the fibrillation process induces disruption to the cellular membrane. We studied the Aβ fibrillation process in two biologically relevant conditions with the peptide either pre-incorporated into or externally added to the synthetic phospholipid bilayers. These two sample preparation conditions mimic the physiological membrane proximities of Aβ peptides before and after the enzymatic cleavage of amyloid precursor protein (APP). Using thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM), we were able to monitor the kinetics and morphological evolution of fibril formation, which was highly sensitive to the two sample preparation protocols. While the external addition protocol generates long and mature fibrils through normal fibrillation process, the pre-incubation protocol was found to stabilize the immature protofibrils. Fluorescence spectroscopy studies with doubly-labeled phospholipids indicated that there may be a lipid uptake process associated with the fibril formation. Solid state nuclear magnetic resonance (NMR) spectroscopy provided evidence for high resolution structural variations in fibrils formed with different protocols, and in particular the stabilization of long-range contact between N- and C-terminal β strands. In addition, disruption of phospholipid bilayers was supported by measurements with ³¹P chemical shifts and relaxation time constants. PMID:24769158

  9. Phospholipid Scramblases

    PubMed Central

    Williamson, Patrick

    2015-01-01

    The distribution of phospholipid types between the two leaflets of a membrane bilayer is a controlled feature of membrane structure. One of the two membrane catalytic activities governing this distribution randomizes the composition of the two leaflets—the phospholipid scramblases. Two proteins (Xkr8 and TMEM16F) required for the activation of these activities have been identified. One of these proteins (TMEM16F) is quite clearly a scramblase itself and provides insight into the mechanism by which transbilayer phospholipid movement is facilitated. PMID:26843813

  10. Effects of chronic ethanol administration and withdrawal on incorporation of arachidonate into membrane phospholipids.

    PubMed

    Sun, G Y; Kelleher, J A; Sun, A Y

    1985-01-01

    A plasma membrane fraction isolated from cerebral cortex of control and ethanol-treated rats was used to study the effects of chronic ethanol administration on uptake of arachidonate by membrane phospholipids. Upon incubation of the membranes with [(14)C] arachidonic acid in the presence of ATP, Mg(2+), and CoA, radioactivity was incorporated into all of the phospholipids, although a large proportion of the label was found in phosphatidylinositols (PI, 60%) and phosphatidylcholines (PC, 20%). Rats given ethanol (8-10 g/kg body wt) via intubation in the form of a liquid diet for 4 weeks showed an increase (17-20%) in arachidonate incorporation into PI and PC as compared to phosphatidylethanolamines (PE) and phosphatidylserines (PS). A similar increase in uptake activity was observed at 2 or 24 h upon withdrawal of ethanol, but uptake activity returned readily to that of control level by 72 h. The method described in this study is a sensitive and reliable procedure for monitoring the arachidonoyl turnover activity in neural membranes with respect to chronic ethanol induction and withdrawal. PMID:20492952

  11. The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study.

    PubMed Central

    Wang, S X; Cai, G P; Sui, S F

    1998-01-01

    Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (pic) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the 'immune cleaning' of plasma liposomes is discussed. PMID:9761718

  12. On the effect of serum on the transport of reactive oxygen species across phospholipid membranes.

    PubMed

    Szili, Endre J; Hong, Sung-Ha; Short, Robert D

    2015-01-01

    The transport of plasma generated reactive oxygen species (ROS) across a simple phospholipid membrane mimic of a (real) cell was investigated. Experiments were performed in cell culture media (Dulbecco's modified Eagle's medium, DMEM), with and without 10% serum. A (broad spectrum) ROS reporter dye, 2,7-dichlorodihydrofluorescein (DCFH), was used to detect the generation of ROS by a helium (He) plasma jet in DMEM using free DCFH and with DCFH encapsulated inside phospholipid membrane vesicles dispersed in DMEM. The authors focus on the concentration and on the relative rates (arbitrary units) for oxidation of DCFH [or the appearance of the oxidized product 2,7-dichlorofluorescein (DCF)] both in solution and within vesicles. In the first 1 h following plasma exposure, the concentration of free DCF in DMEM was ~15× greater in the presence of serum (cf. to the serum-free DMEM control). The DCF in vesicles was ~2× greater in DMEM containing serum compared to the serum-free DMEM control. These data show that serum enhances plasma ROS generation in DMEM. As expected, the role of the phospholipid membrane was to reduce the rate of oxidation of the encapsulated DCFH (with and without serum). And the efficiency of ROS transport into vesicles was lower in DMEM containing serum (at 4% efficiency) when compared to serum-free DMEM (at 32% efficiency). After 1 h, the rate of DCFH oxidation was found to have significantly reduced. Based upon a synthesis of these data with results from the open literature, the authors speculate on how the components of biological fluid and cellular membranes might affect the kinetics of consumption of plasma generated ROS. PMID:25910641

  13. Quantum dots encapsulated within phospholipid membranes: phase-dependent structure, photostability, and site-selective functionalization.

    PubMed

    Zheng, Weiwei; Liu, Yang; West, Ana; Schuler, Erin E; Yehl, Kevin; Dyer, R Brian; Kindt, James T; Salaita, Khalid

    2014-02-01

    Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle-solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle-QD assemblies programmed by Watson-Crick base-pairing. PMID:24417287

  14. Dynamical and Phase Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low Concentration.

    PubMed

    Sharma, V K; Mamontov, E; Tyagi, M; Qian, S; Rai, D K; Urban, V S

    2016-07-01

    The mechanism of action of antimicrobial peptides is traditionally attributed to the formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert a similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature no longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides. PMID:27232190

  15. The tegumental surface membranes of Schistosoma mansoni are enriched in parasite-specific phospholipid species.

    PubMed

    Retra, Kim; deWalick, Saskia; Schmitz, Marion; Yazdanbakhsh, Maria; Tielens, Aloysius G M; Brouwers, Jos F H M; van Hellemond, Jaap J

    2015-08-01

    The complex surface structure of adult Schistosoma mansoni, the tegument, is essential for survival of the parasite. This tegument is syncytial and is covered by two closely-apposed lipid bilayers that form the interactive surface with the host. In order to identify parasite-specific phospholipids present in the tegument, the species compositions of the major glycerophospholipid classes, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, including lysophospholipid species, were analysed in adult S. mansoni worms, isolated tegumental membranes and hamster blood cells. It was shown that there are large differences in species composition in all four phospholipid classes between the membranes of S. mansoni and those of the host blood cells. The species compositions of phosphatidylserine and phosphatidylcholine were strikingly different in the tegument compared with the whole worm. The tegumental membranes are especially enriched in lysophospholipids, predominantly eicosenoic acid (20:1)-containing lyso-phosphatidylserine and lyso-phosphatidylethanolamine species. Furthermore, the tegument was strongly enriched in phosphatidylcholine that contained 5-octadecenoic acid, an unusual fatty acid that is not present in the host. As we have shown previously that lysophospholipids from schistosomes affect the parasite-host interaction, excretion of these tegument-specific phospholipid species was examined in vitro and in vivo. Our experiments demonstrated that these lysophospholipids are not significantly secreted during in vitro incubations and are not detectable in peripheral blood of infected hosts. However, these analyses demonstrated a substantial decrease in PI content of blood plasma from schistosome-infected hamsters, which might indicate that schistosomes influence exosome formation by the host. PMID:25975668

  16. Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties.

    PubMed

    Campbell, R B; Balasubramanian, S V; Straubinger, R M

    2001-05-01

    Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery. PMID:11334622

  17. Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

    PubMed Central

    Ghosh, Manik C.; Ray, Arun K.

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

  18. [Polypeptide and phospholipid composition of the Rickettsia prowazekii membrane and its immunogenic properties].

    PubMed

    Zezerov, E G; Loginov, V S; Berezneva, A S

    1985-06-01

    The composition of the cell membrane in R. prowazekii strain Breinl has been studied by the methods of electrophoresis in 7.5% polyacrylamide gel with 0.1% sodium dodecyl sulfate, iodination with Na125I in the presence of chloramine T or lactoperoxidase and the thin-layer chromatography of the common lipid fraction. Of six major polypeptides contained in whole rickettsial particles (3, 16, 26, 27, 28, 29), five polypeptides (3, 26, 27, 28, 29) making up 54% of all polypeptides of purified rickettsiae have been detected in membrane preparations obtained by either treatment. The molecular weights of these membrane polypeptides are, respectively, 133600, 34000, 29600, 21500 and 12400 daltons. The main membrane phospholipids are phosphatidylethanolamine (68.4%), phosphatidylglycerol (17.2%), phosphatidylcholine (5.1%) and cardiolipin (2.1%). The presence of cholesterol has also been established. The preparations of R. prowazekii membranes and individual membrane polypeptides are immunogenic and induce the formation of specific antibodies in white mice. The preparations of both membranes and surface polypeptide 3 (glycoprotein) have been found to possess a certain protective activity: the effect of the protection of white mice inoculated with R. prowazekii culture has proved to be 64%. PMID:3929506

  19. Drug-Membrane Interactions Studied in Phospholipid Monolayers Adsorbed on Non-porous Alkylated Microspheres

    PubMed Central

    LUKACOVA, VIERA; PENG, MING; FANUCCI, GAIL; TANDLICH, ROMAN; HINDERLITER, ANNE; MAITY, BIKASH; MANIVANNAN, ETHIRAJAN; COOK, GREGORY R.; BALAZ, STEFAN

    2008-01-01

    Characterization of interactions with phospholipids is an integral part of the in vitro profiling of drug candidates because of the roles the interactions play in tissue accumulation and passive diffusion. Currently used test systems may inadequately emulate the bilayer core solvation properties (immobilized artificial membranes - IAM), suffer from potentially slow transport of some chemicals (liposomes in free or immobilized forms), and require a tedious separation (if used for free liposomes). Here we introduce a well-defined system overcoming these drawbacks: nonporous octadecylsilica particles coated with a self-assembled phospholipid monolayer. The coating mimics the structure of the headgroup region, as well as the thickness and properties of the hydrocarbon core more closely than IAM. The monolayer has a similar transition temperature pattern as the corresponding bilayer. The particles can be separated by filtration or a mild centrifugation. The partitioning equilibria of 81 tested chemicals were dissected into the headgroup and core contributions, the latter using the alkane/water partition coefficients. The deconvolution allowed a successful prediction of the bilayer/water partition coefficients with the standard deviation of 0.26 log units. The plate-friendly assay is suitable for high-throughput profiling of drug candidates without sacrificing the quality of analysis or details of the drug-phospholipid interactions. PMID:17218665

  20. [Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the Black Sea fish].

    PubMed

    Silkin, Iu A; Silkina, E N; Zabelinskiĭ, S A

    2012-01-01

    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. PMID:22567974

  1. Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides.

    PubMed

    Mitchell, Natalie J; Seaton, Pamela; Pokorny, Antje

    2016-05-01

    Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains

  2. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  3. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    PubMed

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. PMID:26947183

  4. Probing the Huntingtin 1-17 Membrane Anchor on a Phospholipid Bilayer by Using All-Atom Simulations

    PubMed Central

    Côté, Sébastien; Binette, Vincent; Salnikov, Evgeniy S.; Bechinger, Burkhard; Mousseau, Normand

    2015-01-01

    Mislocalization and aggregation of the huntingtin protein are related to Huntington’s disease. Its first exon—more specifically the first 17 amino acids (Htt17)—is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin’s activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties—order parameter, thickness, and area per lipid—of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions. PMID:25762330

  5. Conformational Transition of Giant DNA in a Confined Space Surrounded by a Phospholipid Membrane

    PubMed Central

    Kato, Ayako; Shindo, Eri; Sakaue, Takahiro; Tsuji, Akihiko; Yoshikawa, Kenichi

    2009-01-01

    It has been established that a long DNA molecule exhibits a large discrete conformational change from a coiled state to a highly folded state in aqueous solution, depending on the presence of various condensing agents such as polyamines. In this study, T4 DNA labeled with fluorescent dyes was encapsulated in a cell-sized microdroplet covered with a phospholipid membrane to investigate the conformational behavior of a DNA molecule in such a confined space. Fluorescence microscopy showed that the presence of Mg2+ induced the adsorption of DNA onto the membrane inner-surface of a droplet composed of phosphatidylethanolamine, while no adsorption was observed onto a phosphatidylcholine membrane. Under the presence of spermine (tetravalent amine), DNA had a folded conformation in the bulk solution. However, when these molecules were encapsulated in the microdroplet, DNA adsorbed onto the membrane surface accompanied by unfolding of its structure into an extended coil conformation under high concentrations of Mg2+. In addition, DNA molecules trapped in large droplets tended not to be adsorbed on the membrane, i.e., no conformational transition occurred. A thermodynamic analysis suggests that the translational entropy loss of a DNA molecule that is accompanied by adsorption is a key factor in these phenomena under micrometer-scale confinement. PMID:19751673

  6. Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.

    PubMed

    Gubbens, Jacob; Ruijter, Eelco; de Fays, Laurence E V; Damen, J Mirjam A; de Kruijff, Ben; Slijper, Monique; Rijkers, Dirk T S; Liskamp, Rob M J; de Kroon, Anton I P M

    2009-01-30

    New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins. PMID:19171301

  7. Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes.

    PubMed

    Moore, D J; Gioioso, S; Sills, R H; Mendelsohn, R

    1999-01-01

    A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes. PMID:9889394

  8. Nonequilibrium patterns of cholesterol-rich chemical heterogenieties within single fluid supported phospholipid bilayer membranes.

    PubMed

    Sapuri-Butti, Annapoorna R; Li, Qijuan; Groves, Jay T; Parikh, Atul N

    2006-06-01

    We have developed a simple method to introduce cholesterol- and sphingomyelin-rich chemical heterogeneities into controlled densities and concentrations within predetermined regions of another distinct fluid phospholipid bilayer supported on a solid substrate. A contiguous primary phase--a fluid POPC bilayer displaying a well-defined array of lipid-free voids (e.g., 20-100 microm squares)--was first prepared on a clean glass surface by microcontact printing under water using a poly(dimethylsiloxane) stamp. The aqueous-phase primary bilayer pattern was subsequently incubated with secondary-phase small unilamellar vesicles composed of independent chemical compositions. Backfilling by comparable vesicles resulted in gradual mixing between the primary- and secondary-phase lipids, effacing the pattern. When the secondary vesicles consisted of phase-separating mixtures of cholesterol, sphingomyelin, and a phospholipid (2:1:1 POPC/sphingomyelin/cholesterol or 1:1:1 DOPC/sphingomyelin/cholesterol), well-defined spatial patterns of fluorescence, chemical compositions, and fluidities emerged. We conjecture that these patterns form because of the differences in the equilibration rates of the secondary liquid-ordered and liquid-disordered phases with the primary fluid POPC phase. The pattern stability depended strongly on the ambient-phase temperature, cholesterol concentration, and miscibility contrast between the two phases. When cholesterol concentration in the secondary vesicles was below 20 mol %, secondary intercalants gradually diffused within the primary POPC bilayer phase, ultimately dissolving the pattern in several minutes and presumably forming a new quasi-equilibrated lipid mixture. These phase domain micropatterns retain some properties of biological rafts including detergent resistance and phase mixing induced by selective cholesterol extraction. These patterns enable direct comparisons of cholesterol- and sphingomyelin-rich phase domains and fluid phospholipid

  9. ATP11C Facilitates Phospholipid Translocation across the Plasma Membrane of All Leukocytes

    PubMed Central

    Yabas, Mehmet; Jing, Weidong; Shafik, Sarah

    2016-01-01

    Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D− (7-AAD−) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD− developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells. PMID:26799398

  10. Selective acceleration of arachidonic acid reincorporation into brain membrane phospholipid following transient ischemia in awake gerbil.

    PubMed

    Rabin, O; Chang, M C; Grange, E; Bell, J; Rapoport, S I; Deutsch, J; Purdon, A D

    1998-01-01

    Awake gerbils were subjected to 5 min of forebrain ischemia by clamping the carotid arteries for 5 min and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 min at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl-CoA pool was independently determined for these fatty acids in control gerbils and following 5 min of ischemia and 5 min of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl-CoAs following recirculation, it was shown that reperfusion after 5 min of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes. PMID:9422378

  11. Liquid crystals and their interactions with colloidal particles and phospholipid membranes: Molecular simulation studies

    NASA Astrophysics Data System (ADS)

    Kim, Evelina B.

    Experimentally, liquid crystals (LC) can be used as the basis for optical biomolecular sensors that rely on LC ordering. Recently, the use of LC as a reporting medium has been extended to investigations of molecular scale processes at lipid laden aqueous-LC interfaces and at biological cell membranes. In this thesis, we present two related studies where liquid crystals are modelled at different length scales. We examine (a) the behavior of nanoscopic colloidal particles in LC systems, using Monte Carlo (MC) molecular simulations and a mesoscopic dynamic field theory (DyFT); and (b) specific interactions of two types of mesogens with a model phospholipid bilayer, using atomistic molecular dynamics (MD) at the A-nm scale. In (a), we consider colloidal particles suspended in a LC, confined between two walls. We calculate the colloid-substrate and colloid-colloid potentials of mean force (PMF). For the MC simulations, we developed a new technique (ExEDOS or Expanded Ensemble Density Of States) that ensures good sampling of phase space without prior knowledge of the energy landscape of the system. Both results, simulation and DyFT, indicate a repulsive force acting between a colloid and a wall. In contrast, both techniques indicate an overall colloid-colloid attraction and predict a new topology of the disclination lines that arises when the particles approach each other. In (b), we find that mesogens (pentylcyanobiphenyl [5CB] or difluorophenyl-pentylbicyclohexyl [5CF]) preferentially partition from the aqueous phase into a dipalmitoylphosphatidylcholine (DPPC) bilayer. We find highly favorable free energy differences for partitioning (-18kBT for 5CB, -26k BT for 5CF). We also simulated fully hydrated bilayers with embedded 5CB or 5CF at concentrations used in recent experiments (6 mol% and 20 mol%). The presence of mesogens in the bilayer enhances the order of lipid acyl tails and changes the spatial and orientational arrangement of lipid headgroup atoms. A stronger

  12. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

    PubMed Central

    Mouchlis, Varnavas D.; Bucher, Denis; McCammon, J. Andrew; Dennis, Edward A.

    2015-01-01

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface. PMID:25624474

  13. ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

    PubMed Central

    Seigneuret, M; Devaux, P F

    1984-01-01

    Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape. Images PMID:6587389

  14. Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

    PubMed

    Randall, Alex S; Liu, Che-Hsiung; Chu, Brian; Zhang, Qifeng; Dongre, Sidharta A; Juusola, Mikko; Franze, Kristian; Wakelam, Michael J O; Hardie, Roger C

    2015-02-11

    Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade. PMID:25673862

  15. Speed and Sensitivity of Phototransduction in Drosophila Depend on Degree of Saturation of Membrane Phospholipids

    PubMed Central

    Randall, Alex S.; Liu, Che-Hsiung; Chu, Brian; Zhang, Qifeng; Dongre, Sidharta A.; Juusola, Mikko; Franze, Kristian; Wakelam, Michael J.O.

    2015-01-01

    Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca2+ influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade. PMID:25673862

  16. A phospholipid-apolipoproteinA-I nanoparticle containing Amphotericin B as a drug delivery platform with cell membrane protective properties

    PubMed Central

    Burgess, Braydon L.; Cavigiolio, Giorgio; Fannucchi, Michelle V.; Illek, Beate; Forte, Trudy M.; Oda, Michael N.

    2010-01-01

    Amphotericin B (AMB), a potent antifungal agent, has been employed as an inhalable therapy for pulmonary fungal infections. We recently described a novel nano-sized delivery vehicle composed of phospholipid (PL) and apolipoprotein A-I, NanoDisk (ND), to which we added AMB as a payload (ND-AMB). The goal of the present study was to evaluate whether ND-AMB, compared to other formulations, preserves lung cell integrity in vitro, as AMB can be toxic to mammalian cells and reduce lung function when inhaled. Epithelial integrity was assessed by measuring K+ ion flux across a model airway epithelium, Calu-3 cells. In this assay ND-AMB was at least 8-fold less disruptive than AMB/deoxycholate (DOC). Cell viability studies confirmed this observation. Unexpectedly, the ND vehicle restored the integrity of a membrane compromised by prior exposure to AMB. An alternative formulation of ND-AMB containing a high load of AMB per ND was not protective, suggesting that ND with a low ratio of AMB to PL can sequester additional AMB from membranes. ND-AMB also protected HepG2 cells from the cytotoxicity of AMB, as determined by cellular viability and lactate dehydrogenase (LDH) levels. This study suggests that ND-AMB may be safe for administration via inhalation and reveals a unique activity whereby ND-AMB protects lung epithelial membranes from AMB toxicity. PMID:20696226

  17. Adaptive modification of membrane phospholipid fatty acid composition and metabolic thermosuppression of brown adipose tissue in heat-acclimated rats

    NASA Astrophysics Data System (ADS)

    Saha, S. K.; Ohno, T.; Tsuchiya, K.; Kuroshima, A.

    Thermogenesis, especially facultative thermogenesis by brown adipose tissue (BAT), is less important in high ambient temperature and the heat-acclimated animals show a lower metabolic rate. Adaptive changes in the metabolic activity of BAT are generally found to be associated with a modification of membrane phospholipid fatty acid composition. However, the effect of heat acclimation on membrane phospholipid fatty acid composition is as yet unknown. In this study, we examined the thermogenic activity and phospholipid fatty acid composition of interscapular BAT from heat-acclimated rats (control: 25+/-1°C, 50% relative humidity and heat acclimation: 32+/-0.5°C, 50% relative humidity). Basal thermogenesis and the total thermogenic capacity after noradrenaline stimulation, as estimated by in vitro oxygen consumption of BAT (measured polarographically using about 1-mm3 tissue blocks), were smaller in the heat-acclimated group than in the control group. There was no difference in the tissue content of phospholipids between the groups when expressed per microgram of DNA. The phospholipid fatty acid composition was analyzed by a capillary gas chromatograph. The state of phospholipid unsaturation, as estimated by the number of double bonds per fatty acid molecule, was similar between the groups. The saturated fatty acid level was higher in the heat-acclimated group. Among the unsaturated fatty acids, heat acclimation decreased docosahexaenoic acid and oleic acid levels, and increased the arachidonic acid level. The tissue level of docosahexaenoic acid correlated with the basal oxygen consumption of BAT (r=0.6, P<0.01) and noradrenaline-stimulated maximum values of oxygen consumption (r=0.5, P<0.05). Our results show that heat acclimation modifies the BAT phospholipid fatty acids, especially the n-3 polyunsaturated fatty acid docosahexaenoic acid, which is possibly involved in the metabolic thermosuppression.

  18. Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

    SciTech Connect

    Paliyath, G.; Thompson, J.E.

    1987-01-01

    Evidence for the involvement of Ca/sup 2 +/ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from (U-/sup 14/C)phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of (U-/sup 14/C) phosphatidylcholine, viz. phospholipase D phosphatidic acid phosphatase and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca/sup 2 +/, whereas lipolytic acyl hydrolase proved to be insensitive to Ca/sup 2 +/. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca/sup 2 +/. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC/sub 50/ values ranging from 10 to 15 micromolar. Thus, the Ca/sup 2 +/-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca/sup 2 +/ on phospholipase D is independent of calmodulin. The role of Ca/sup 2 +/ as a second messenger in the initiation of membrane lipid degradation is discussed.

  19. Co-existence of Gel and Fluid Lipid Domains in Single-component Phospholipid Membranes

    SciTech Connect

    Armstrong, Clare L; Barrett, M; Toppozini, L; Yamani, Zahra; Kucerka, Norbert; Katsaras, John; Fragneto, Giovanna; Rheinstadter, Maikel C

    2012-01-01

    Lateral nanostructures in membranes, so-called rafts, are believed to strongly influence membrane properties and functions. The experimental observation of rafts has proven difficult as they are thought to be dynamic structures that likely fluctuate on nano- to microsecond time scales. Using neutron diffraction we present direct experimental evidence for the co-existence of gel and fluid lipid domains in a single-component phospholipid membrane made of DPPC as it undergoes its main phase transition. The coherence length of the neutron beam sets a lower limit for the size of structures that can be observed. Neutron coherence lengths between 30 and 242A used in this study were obtained by varying the incident neutron energy and the resolution of the neutron spectrometer. We observe Bragg peaks corresponding to co-existing nanometer sized structures, both in out-of-plane and in-plane scans, by tuning the neutron coherence length. During the main phase transition, instead of a continuous transition that shows a pseudo-critical behavior, we observe the co-existence of gel and fluid domains.

  20. Efficient photoinduced orthogonal energy and electron transfer reactions via phospholipid membrane-bound donors and acceptors

    SciTech Connect

    Clapp, P.J.; Armitage, B.; Roosa, P.; O'Brien, D.F. )

    1994-10-05

    A three component, liposome-bound photochemical molecular device (PMD) consisting of energy and electron transfer reactions is described. Bilayer membrane surface-associated dyes, 5,10,15,20-tetrakis[4-(trimethylammonio)-phenyl]-21H,2 3H-porphine tetra-p-tosylate salt and N,N[prime]-bis[(3-trimethylammonio)propyl]thiadicarbocya nine tribromide, are the energy donor and acceptor, respectively, in a blue light stimulated energy transfer reaction along the vesicle surface. The electronically excited cyanine is quenched by electron transfer from the phospholipid membrane bound triphenylbenzyl borate anion, which is located in the lipid bilayer interior. The PMD exhibits sequential reactions following electronic excitation with the novel feature that the steps proceed with orthogonal orientation: energy transfer occurs parallel to the membrane surface, and electron transfer occurs perpendicular to the surface. Photobleaching and fluorescence quenching experiments verify the transfer reactions, and Stern-Volmer analysis was used to estimate the reaction rate constants. At the highest concentrations examined of energy and electron acceptor ca. 60% of the photoexcited porphyrins were quenched by energy transfer to the cyanine. 56 refs., 6 figs., 3 tabs.

  1. Dynamical and phase behavior of a phospholipid membrane altered by an antimicrobial peptide at low concentration

    DOE PAGESBeta

    Mamontov, Eugene; Tyagi, M.; Qian, Shuo; Rai, Durgesh K.; Urban, Volker S.; Sharma, V. K.

    2016-05-27

    Here we discuss that the mechanism of action of antimicrobial peptides is traditionally attributed to the formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert a similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature nomore » longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides.« less

  2. Adaptation of the bacterial membrane to changing environments using aminoacylated phospholipids

    PubMed Central

    Roy, Hervé; Dare, Kiley; Ibba, Michael

    2009-01-01

    Summary Fine-tuning of the biophysical properties of biological membranes is essential for adaptation of cells to changing environments. For instance, to lower the negative charge of the lipid bilayer, certain bacteria add lysine to phosphatidylglycerol (PG) converting the net negative charge of PG (–1) to a net positive charge in Lys-PG (+1). Reducing the net negative charge of the bacterial cell wall is a common strategy used by bacteria to resist cationic antimicrobial peptides (CAMPs) secreted by other microbes or produced by the innate immune system of a host organism. The article by Klein et al. in the current issue of Molecular Microbiology reports a new modification of the bacterial membrane, addition of alanine to PG, in Pseudomonas aeruginosa. In spite of the neutral charge of Ala-PG, this modified lipid was found to be linked to several resistance phenotypes in P. aeruginosa. For instance, Ala-PG increases resistance to two positively charged antibacterial agents, a β-lactam and high concentrations of lactate. These findings shed light on the mechanisms by which bacteria fine-tune the properties of their cell membranes by adding various amino acids on the polar head group of phospholipids. PMID:19054327

  3. Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers

    NASA Astrophysics Data System (ADS)

    Lu, George J.; Park, Sang Ho; Opella, Stanley J.

    2012-07-01

    We demonstrate 1H amide resonance line widths <300 Hz in 1H/15N heteronuclear correlation (HETCOR) spectra of membrane proteins in aligned phospholipid bilayers. This represents a substantial improvement over typically observed line widths of ˜1 kHz. Furthermore, in a proton detected local field (PDLF) version of the experiment that measures heteronuclear dipolar couplings, line widths <130 Hz are observed. This dramatic line narrowing of 1H amide resonances enables many more individual signals to be resolved and assigned from uniformly 15N labeled membrane proteins in phospholipid bilayers under physiological conditions of temperature and pH. Finding that the decrease in line widths occurs only for membrane proteins that undergo fast rotational diffusion around the bilayer normal, but not immobile molecules, such as peptide single crystals, identifies a potential new direction for pulse sequence development that includes overall molecular dynamics in their design.

  4. Generation, modulation and maintenance of the plasma membrane asymmetric phospholipid composition in yeast cells during growth: their relation to surface potential and membrane protein activity.

    PubMed

    Cerbón, J; Calderón, V

    1995-04-12

    During growth a cyclic exposure of anionic phospholipids to the external surface of the plasma membrane was found. The surface charge density (sigma) increased gradually reaching a maximum in the first 5 h of growth and returned gradually to their initial value at the end of the logarithmic phase of growth (10-12 h). Phosphatidylinositol, that determines to a large extent the magnitude of the sigma, increased 83% in the yeast cells during the first 4 h of growth and returned gradually to their initial level at 10-12 h. During the stationary phase (12-24 h), both sigma and the anionic/zwitterionic phospholipid ratio, remained without any significant variation. The high-affinity H-linked glutamate transport system that behaves as a sensor of the changes in the membrane surface potential (phi) increased its activity in the first 5 h and then decreased it, following with great accuracy the sigma variations and remained without changes during the stationary phase of growth. The phosphatidylserine (PS) relative concentration in the cells (9.0%) did not significantly change during the whole growth curve, but their asymmetric distribution varied, contributing to the changes in sigma. PS facing the outer membrane surface increased 2.45-times during the first 5 h of growth and then returned to their original value at the end of the log phase (12 h). Phosphatidylcholine (PC) remained constant during the whole growth curve (50%), while phosphatidylethanolamine (PE) decreased 3-fold in the first 4 h and then increased to its original value at 10 h. Interestingly, PE at the outer membrane surface remained constant (3% of the total phospholipids) during the whole growth curve. During growth yeast cells change their phospholipid composition originating altered patterns of the plasma membrane phospholipid composition and IN-OUT distribution. This dynamic asymmetry is involved in the regulation of the surface potential and membrane protein activity. PMID:7718598

  5. Improvement of pharmacokinetic and antitumor activity of layered double hydroxide nanoparticles by coating with PEGylated phospholipid membrane

    PubMed Central

    Yan, Mina; Zhang, Zhaoguo; Cui, Shengmiao; Lei, Ming; Zeng, Ke; Liao, Yunhui; Chu, Weijing; Deng, Yihui; Zhao, Chunshun

    2014-01-01

    Layered double hydroxide (LDH) has attracted considerable attention as a drug carrier. However, because of its poor in vivo behavior, polyethylene glycolylated (PEGylated) phospholipid must be used as a coformer to produce self-assembled core–shell nanoparticles. In the present study, we prepared a PEGylated phospholipid-coated LDH (PLDH) (PEG-PLDH) delivery system. The PEG-PLDH nanoparticles had an average size of 133.2 nm. Their core–shell structure was confirmed by transmission electron microscopy and X-ray photoelectron spectroscopy. In vitro liposome-cell-association and cytotoxicity experiments demonstrated its ability to be internalized by cells. In vivo studies showed that PEGylated phospholipid membranes greatly reduced the blood clearance rate of LDH nanoparticles. PEG-PLDH nanoparticles demonstrated a good control of tumor growth and increased the survival rate of mice. These results suggest that PEG-PLDH nanoparticles can be a useful drug delivery system for cancer therapy. PMID:25364245

  6. A class of supported membranes: formation of fluid phospholipid bilayers on photonic band gap colloidal crystals.

    PubMed

    Brozell, Adrian M; Muha, Michelle A; Sanii, Babak; Parikh, Atul N

    2006-01-11

    We report the formation of a new class of supported membranes consisting of a fluid phospholipid bilayer coupled directly to a broadly tunable colloidal crystal with a well-defined photonic band gap. For nanoscale colloidal crystals exhibiting a band gap at the optical frequencies, substrate-induced vesicle fusion gives rise to a surface bilayer riding onto the crystal surface. The bilayer is two-dimensionally continuous, spanning multiple beads with lateral mobilities which reflect the coupling between the bilayer topography and the curvature of the supporting colloidal surface. In contrast, the spreading of vesicles on micrometer scale colloidal crystals results in the formation of bilayers wrapping individual colloidal beads. We show that simple UV photolithography of colloidal crystals produces binary patterns of crystal wettabilities, photonic stopbands, and corresponding patterns of lipid mono- and bilayer morphologies. We envisage that these approaches will be exploitable for the development of optical transduction assays and microarrays for many membrane-mediated processes, including transport and receptor-ligand interactions. PMID:16390122

  7. Characterization of Physical Properties of Supported Phospholipid Membranes Using Imaging Ellipsometry at Optical Wavelengths

    PubMed Central

    Howland, Michael C.; Szmodis, Alan W.; Sanii, Babak; Parikh, Atul N.

    2007-01-01

    Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids. PMID:17142265

  8. Phospholipid base exchange activity in the leukocyte membranes of patients with inflammatory disorders.

    PubMed Central

    Niwa, Y.; Sakane, T.; Ozaki, Y.; Kanoh, T.; Taniguchi, S.

    1987-01-01

    Phospholipid base exchange and cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) activities were assessed in the membranes of neutrophils or lymphocytes from patients with various inflammatory disorders. Ethanolamine exchange activity was significantly enhanced in both neutrophils and lymphocytes from patients with active Behçet's disease, active systemic lupus erythematosus (SLE), and severe bacterial infections and slightly enhanced in those from patients with active rheumatoid arthritis (RA), compared with healthy controls. No abnormal findings were found in CPT, EPT, or serine or choline base exchange activities in the leukocytes from any of the diseased groups tested or in the ethanolamine exchange activity of patients with severe viral infections and inactive SLE, RA, and Behçet's disease. The authors have recently demonstrated the enhancement of transmethylation and phospholipase A2 activity in human leukocyte membranes at the height of inflammatory disease states, as well as the activation of leukocyte ethanolamine exchange by bioactive stimulants. These data postulate that phosphatidylethanolamine synthesis by the base exchange reaction may be the precursor of transmethylation and its subsequent activation of phospholipase A2, leading to the induction of arachidonic acid cascade. PMID:3034067

  9. Structure Determination of a Membrane Protein with Two Trans-membrane Helices in Aligned Phospholipid Bicelles by Solid-state NMR Spectroscopy

    PubMed Central

    De Angelis, Anna A.; Howell, Stanley C.; Nevzorov, Alexander A.; Opella, Stanley J.

    2011-01-01

    The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively 15N labeled samples to resolve and assign the backbone amide resonances, and to measure the associated 15N chemical shift and 1H-15N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. 1H/13C/15N triple-resonance experiments were applied to selectively 13C′ and 15N labeled samples to complete the resonance assignments, especially for residues in the non-helical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers. PMID:16967977

  10. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    SciTech Connect

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A.

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  11. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers

    PubMed Central

    2004-01-01

    CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP–Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP–Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway. PMID:15270716

  12. Binding of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein (MRP) to vesicular phospholipid membranes.

    PubMed Central

    Vergères, G; Ramsden, J J

    1998-01-01

    The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions. PMID:9461483

  13. Diffusion in phospholipid bilayer membranes: dual-leaflet dynamics and the roles of tracer–leaflet and inter-leaflet coupling

    PubMed Central

    Hill, Reghan J.; Wang, Chih-Ying

    2014-01-01

    A variety of observations—sometimes controversial—have been made in recent decades when attempting to elucidate the roles of interfacial slip on tracer diffusion in phospholipid membranes. Evans–Sackmann theory (1988) has furnished membrane viscosities and lubrication-film thicknesses for supported membranes from experimentally measured lateral diffusion coefficients. Similar to the Saffman and Delbrück model, which is the well-known counterpart for freely supported membranes, the bilayer is modelled as a single two-dimensional fluid. However, the Evans–Sackman model cannot interpret the mobilities of monotopic tracers, such as individual lipids or rigidly bound lipid assemblies; neither does it account for tracer–leaflet and inter-leaflet slip. To address these limitations, we solve the model of Wang and Hill, in which two leaflets of a bilayer membrane, a circular tracer and supports are coupled by interfacial friction, using phenomenological friction/slip coefficients. This furnishes an exact solution that can be readily adopted to interpret the mobilities of a variety of mosaic elements—including lipids, integral monotopic and polytopic proteins, and lipid rafts—in supported bilayer membranes. PMID:25002822

  14. Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

    PubMed

    Fernández-Carvajal, Asia M; Encinar, José A; Poveda, José Antonio; de Juan, Entilio; Martínez-Pinna, Juan; Ivorra, Isabel; Ferragut, José Antonio; Morales, Andrés; González-Ros, José Manuel

    2006-01-01

    Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of

  15. Interactions of amelogenin with phospholipids.

    PubMed

    Lokappa, Sowmya Bekshe; Chandrababu, Karthik Balakrishna; Dutta, Kaushik; Perovic, Iva; Evans, John Spencer; Moradian-Oldak, Janet

    2015-02-01

    Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. PMID:25298002

  16. Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.

    PubMed

    Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J O; Bakal, Chris

    2015-09-01

    We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. PMID:26333836

  17. Multi-scale molecular dynamics study of cholera pentamer binding to a GM1-phospholipid membrane.

    PubMed

    Sridhar, Akshay; Kumar, Amit; Dasmahapatra, Ashok Kumar

    2016-07-01

    The AB5 type toxin produced by the Vibrio cholerae bacterium is the causative agent of the cholera disease. The cholera toxin (CT) has been shown to bind specifically to GM1 glycolipids on the membrane surface. This binding of CT to the membrane is the initial step in its endocytosis and has been postulated to cause significant disruption to the membrane structure. In this work, we have carried out a combination of coarse-grain and atomistic simulations to study the binding of CT to a membrane modelled as an asymmetrical GM1-DPPC bilayer. Simulation results indicate that the toxin binds to the membrane through only three of its five B subunits, in effect resulting in a tilted bound configuration. Additionally, the binding of the CT can increase the area per lipid of GM1 leaflet, which in turn can cause the membrane regions interacting with the bound subunits to experience significant bilayer thinning and lipid tail disorder across both the leaflets. PMID:27474868

  18. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    PubMed Central

    Roche, Julien; Louis, John M.; Aniana, Annie; Ghirlando, Rodolfo; Bax, Ad

    2015-01-01

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion. PMID:25631354

  19. Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein.

    PubMed

    Manojlovic, V; Winkler, K; Bunjes, V; Neub, A; Schubert, R; Bugarski, B; Leneweit, G

    2008-07-15

    Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron

  20. A silybin-phospholipid complex prevents mitochondrial dysfunction in a rodent model of nonalcoholic steatohepatitis.

    PubMed

    Serviddio, Gaetano; Bellanti, Francesco; Giudetti, Anna Maria; Gnoni, Gabriele Vincenzo; Petrella, Antonio; Tamborra, Rosanna; Romano, Antonino Davide; Rollo, Tiziana; Vendemiale, Gianluigi; Altomare, Emanuele

    2010-03-01

    Mitochondrial dysfunction and oxidative stress are determinant events in the pathogenesis of nonalcoholic steatohepatitis. Silybin has shown antioxidant, anti-inflammatory, and antifibrotic effects in chronic liver disease. We aimed to study the effect of the silybin-phospholipid complex (SILIPHOS) on liver redox balance and mitochondrial function in a dietary model of nonalcoholic steatohepatitis. To accomplish this, glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H(2)O(2) production rate were evaluated in isolated liver mitochondria from rats fed a methionine- and choline-deficient (MCD) diet and the MCD diet plus SILIPHOS for 7 and 14 weeks. Oxidative proteins, hydroxynonenal (HNE)- and malondialdehyde (MDA)-protein adducts, and mitochondrial membrane lipid composition were also measured. Treatment with SILIPHOS limited glutathione depletion and mitochondrial H(2)O(2) production. Moreover, SILIPHOS preserved mitochondrial bioenergetics and prevented mitochondrial proton leak and ATP reduction. Finally, SILIPHOS limited the formation of HNE- and MDA-protein adducts. In conclusion, SILIPHOS is effective in preventing severe oxidative stress and preserving hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis induced by the MCD diet. The modifications of mitochondrial membrane fatty acid composition induced by the MCD diet are partially prevented by SILIPHOS, conferring anti-inflammatory and antifibrotic effects. The increased vulnerability of lipid membranes to oxidative damage is limited by SILIPHOS through preserved mitochondrial function. PMID:20008062

  1. A carbon-13 nuclear magnetic resonance spectroscopic study of inter-proton pair order parameters: a new approach to study order and dynamics in phospholipid membrane systems.

    PubMed

    Urbina, J A; Moreno, B; Arnold, W; Taron, C H; Orlean, P; Oldfield, E

    1998-09-01

    We report a simple new nuclear magnetic resonance (NMR) spectroscopic method to investigate order and dynamics in phospholipids in which inter-proton pair order parameters are derived by using high resolution 13C cross-polarization/magic angle spinning (CP/MAS) NMR combined with 1H dipolar echo preparation. The resulting two-dimensional NMR spectra permit determination of the motionally averaged interpair second moment for protons attached to each resolved 13C site, from which the corresponding interpair order parameters can be deducted. A spin-lock mixing pulse before cross-polarization enables the detection of spin diffusion amongst the different regions of the lipid molecules. The method was applied to a variety of model membrane systems, including 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/sterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sterol model membranes. The results agree well with previous studies using specifically deuterium labeled or predeuterated phospholipid molecules. It was also found that efficient spin diffusion takes place within the phospholipid acyl chains, and between the glycerol backbone and choline headgroup of these molecules. The experiment was also applied to biosynthetically 13C-labeled ergosterol incorporated into phosphatidylcholine bilayers. These results indicate highly restricted motions of both the sterol nucleus and the aliphatic side chain, and efficient spin exchange between these structurally dissimilar regions of the sterol molecule. Finally, studies were carried out in the lamellar liquid crystalline (L alpha) and inverted hexagonal (HII) phases of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These results indicated that phosphatidylethanolamine lamellar phases are more ordered than the equivalent phases of phosphatidylcholines. In the HII (inverted hexagonal) phase, despite the increased translational freedom, there is highly constrained packing of the lipid molecules, particularly in

  2. Investigating the protective properties of milk phospholipids against ultraviolet light exposure in a skin equivalent model

    NASA Astrophysics Data System (ADS)

    Russell, Ashley; Laubscher, Andrea; Jimenez-Flores, Rafael; Laiho, Lily H.

    2010-02-01

    Current research on bioactive molecules in milk has documented health advantages of bovine milk and its components. Milk Phospholipids, selected for this study, represent molecules with great potential benefit in human health and nutrition. In this study we used confocal reflectance and multiphoton microscopy to monitor changes in skin morphology upon skin exposure to ultraviolet light and evaluate the potential of milk phospholipids in preventing photodamage to skin equivalent models. The results suggest that milk phospholipids act upon skin cells in a protective manner against the effect of ultraviolet (UV) radiation. Similar results were obtained from MTT tissue viability assay and histology.

  3. The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders

    PubMed Central

    2012-01-01

    Gastrointestinal symptoms and altered blood phospholipid profiles have been reported in patients with autism spectrum disorders (ASD). Most of the phospholipid analyses have been conducted on the fatty acid composition of isolated phospholipid classes following hydrolysis. A paucity of information exists on how the intact phospholipid molecular species are altered in ASD. We applied ESI/MS to determine how brain and blood intact phospholipid species were altered during the induction of ASD-like behaviors in rats following intraventricular infusions with the enteric bacterial metabolite propionic acid. Animals were infused daily for 8 days, locomotor activity assessed, and animals killed during the induced behaviors. Propionic acid infusions increased locomotor activity. Lipid analysis revealed treatment altered 21 brain and 30 blood phospholipid molecular species. Notable alterations were observed in the composition of brain SM, diacyl mono and polyunsaturated PC, PI, PS, PE, and plasmalogen PC and PE molecular species. These alterations suggest that the propionic acid rat model is a useful tool to study aberrations in lipid metabolism known to affect membrane fluidity, peroxisomal function, gap junction coupling capacity, signaling, and neuroinflammation, all of which may be associated with the pathogenesis of ASD. PMID:22747852

  4. Interaction of three-finger toxins with phospholipid membranes: comparison of S- and P-type cytotoxins

    PubMed Central

    2004-01-01

    The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of Naja oxiana (the Central Asian cobra). They belong to S- and P-type respectively based on Ser-28 and Pro-30 residues within a putative phospholipid bilayer binding site. Previously, we investigated the interaction of CTII with multilamellar liposomes of dipalmitoylphosphatidylglycerol by wide-line 31P-NMR spectroscopy. To compare interactions of these proteins with phospholipids, we investigated the interaction of CTI with the multilamellar liposomes of dipalmitoylphosphatidylglycerol analogously. The effect of CTI on the chemical shielding anisotropy and deformation of the liposomes in the magnetic field was determined at different temperatures and lipid/protein ratios. It was found that both the proteins do not affect lipid organization in the gel state. In the liquid crystalline state of the bilayer they disturb lipid packing. To get insight into the interactions of the toxins with membranes, Monte Carlo simulations of CTI and CTII in the presence of the bilayer membrane were performed. It was found that both the toxins penetrate into the bilayer with the tips of all the three loops. However, the free-energy gain on membrane insertion of CTI is smaller (by ≈7 kcal/mol; 1 kcal≡4.184 kJ) when compared with CTII, because of the lower hydrophobicity of the membrane-binding site of CTI. These results clearly demonstrate that the P-type cytotoxins interact with membranes stronger than those of the S-type, although the mode of the membrane insertion is similar for both the types. PMID:15584897

  5. Effect of Selection for High Activity-Related Metabolism on Membrane Phospholipid Fatty Acid Composition in Bank Voles.

    PubMed

    Stawski, Clare; Valencak, Teresa G; Ruf, Thomas; Sadowska, Edyta T; Dheyongera, Geoffrey; Rudolf, Agata; Maiti, Uttaran; Koteja, Paweł

    2015-01-01

    Endothermy, high basal metabolic rates (BMRs), and high locomotor-related metabolism were important steps in the evolution of mammals. It has been proposed that the composition of membrane phospholipid fatty acids plays an important role in energy metabolism and exercise muscle physiology. In particular, the membrane pacemaker theory of metabolism suggests that an increase in cell membrane fatty acid unsaturation would result in an increase in BMR. We aimed to determine whether membrane phospholipid fatty acid composition of heart, liver, and gastrocnemius muscles differed between lines of bank voles selected for high swim-induced aerobic metabolism-which also evolved an increased BMR-and unselected control lines. Proportions of fatty acids significantly differed among the organs: liver was the least unsaturated, whereas the gastrocnemius muscles were most unsaturated. However, fatty acid proportions of the heart and liver did not differ significantly between selected and control lines. In gastrocnemius muscles, significant differences between selection directions were found: compared to control lines, membranes of selected voles were richer in saturated C18:0 and unsaturated C18:2n-6 and C18:3n-3, whereas the pattern was reversed for saturated C16:0 and unsaturated C20:4n-6. Neither unsaturation index nor other combined indexes of fatty acid proportions differed between lines. Thus, our results do not support the membrane pacemaker hypothesis. However, the differences between selected and control lines in gastrocnemius muscles reflect chain lengths rather than number of double bonds and are probably related to differences in locomotor activity per se rather than to differences in the basal or routine metabolic rate. PMID:26658414

  6. Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress

    PubMed Central

    Liu, Xiao-Yu; Ouyang, Long-Ling; Zhou, Zhi-Gang

    2016-01-01

    In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress. PMID:27216435

  7. Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress.

    PubMed

    Liu, Xiao-Yu; Ouyang, Long-Ling; Zhou, Zhi-Gang

    2016-01-01

    In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress. PMID:27216435

  8. Swelling of phospholipid membranes by divalent metal ions depends on the location of the ions in the bilayers.

    PubMed

    Alsop, Richard J; Maria Schober, Rafaëla; Rheinstädter, Maikel C

    2016-08-10

    The Hofmeister series illustrates how salts produce a wide range of effects in biological systems, which are not exclusively explained by ion charge. In lipid membranes, charged ions have been shown to bind to lipids and either hydrate or dehydrate lipid head groups, and also to swell the water layer in multi-lamellar systems. Typically, Hofmeister phenomena are explained by the interaction of the ions with water, as well as with biological interfaces, such as proteins or membranes. We studied the effect of the divalent cations Mg(2+), Ca(2+), Fe(2+), and Zn(2+) on oriented, stacked, phospholipid bilayers made of dimyristoylphosphatidylcholine (DMPC). Using high-resolution X-ray diffraction, we observed that the cations lead to a swelling of the water layer between the bilayers, without causing significant changes to the bilayer structure. The cations swelled the bilayers in different amounts, in the order Fe(2+) > Mg(2+) > Ca(2+) > Zn(2+). By decomposing the total bilayer electron density into different molecular groups, Zn(2+) and Ca(2+) were found to interact with the glycerol groups of the lipid molecules and cause minor swelling of the bilayers. Mg(2+) and Fe(2+) were found to position near the phosphate groups and cause a strong increase in the number of hydration water molecules. Our results present a molecular mechanism-of-action for the Hofmeister series in phospholipid membranes. PMID:27453289

  9. Design and synthesis of basic peptides having amphipathic beta-structure and their interaction with phospholipid membranes.

    PubMed

    Ono, S; Lee, S; Mihara, H; Aoyagi, H; Kato, T; Yamasaki, N

    1990-02-28

    Basic amphipathic beta-structural peptides, Ac-(Ser-Val-Lys-Val)n-NHCH3 (1n, n = 1-3) and Ac-(Lys-Val)n-NHCH3 (2n, n = 2-4), were synthesized and their interaction with DPPC and DPPC-DPPG (3:1) bilayers was studied by CD, dye-leakage and fluorescence experiments. The CD data indicated that oligopeptides consisting of more than eight residues with alternating hydrophobic (Val) and hydrophilic amino acids (Ser and Lys) were able to form an amphipathic beta-structure in acidic phospholipid bilayers, but not or weakly in aqueous solution and in neutral phospholipid bilayers. The dye-leakage experiment showed that the basic amphipathic beta-structural peptides interact with acidic phospholipid bilayers to perturb them, but less effectively compared with basic amphipathic alpha-helical peptides. Fluorescent spectroscopic data suggest that hydrophobic side of the amphipathic peptides may immerse into membrane without deep penetration. Based on these results, we postulate that the formation of the basic amphipathic beta-structure on acidic lipid bilayers may be due to the combined effect of electrostatic and hydrophobic interactions between basic peptides and acidic lipid bilayers. PMID:2306456

  10. Molecular and structural composition of phospholipid membranes in livers of marine and freshwater fish in relation to temperature.

    PubMed Central

    Dey, I; Buda, C; Wiik, T; Halver, J E; Farkas, T

    1993-01-01

    The compositions and physical states of the liver phospholipids of marine and freshwater fish adapted to relatively constant but radically different temperatures were investigated. Fish adapted to low temperature (5-10 degrees C) accumulated more unsaturated fatty acids than those in a warm (25-27 degrees C) environment. There were no measurable differences in the gross fatty acid compositions of the total liver phospholipids from identical thermal environments. Docosahexaenoic acid (22:6) did not seem to participate in the process of adaptation. Cold adaptation was coincidental with oleic acid (18:1) accumulation, preferentially in the phosphatidylethanolamine. Determination of the molecular species composition of phosphatidylethanolamine revealed a 2- to 3-fold and 10-fold increase in the level of 18:1/22:6 and 18:1/20:5 species, respectively. ESR spectroscopy revealed a 7-10% compensation in the ordering state of native phospholipids with temperature. Combination of 16:0/22:6 phosphatidylcholine with phosphatidylethanolamines of cold-adapted marine fish showed a drastic fluidization near the C-2 segment of the bilayer, but not in the deeper regions. An appropriate combination (75:25) of phosphatidylcholines from warmth-adapted marine fish with phosphatidylethanolamines from cold-adapted marine fish mimicked a 100% adaptational efficacy in the C-2 segment as compared with the phosphatidylethanolamines of warmth-adapted marine fish. A specific role of 18:1/22:6 phosphatidylethanolamine in controlling membrane structure and physical state with thermal adaptation is proposed. PMID:8356045

  11. Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles.

    PubMed

    Valpuesta, J M; Arrondo, J L; Barbero, M C; Pons, M; Goñi, F M

    1986-05-15

    Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The

  12. Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

    SciTech Connect

    Valpuesta, J.M.; Arrondo, J.L.; Barbero, M.C.; Pons, M.; Goni, F.M.

    1986-05-15

    Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear.

  13. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients. PMID:26907692

  14. The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.

    PubMed

    Salvioli, Rosa; Tatti, Massimo; Scarpa, Susanna; Moavero, Sabrina Maria; Ciaffoni, Fiorella; Felicetti, Federica; Kaneski, Christine R; Brady, Roscoe O; Vaccaro, Anna Maria

    2005-08-15

    The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (approximately 20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo. PMID:15826241

  15. A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles.

    PubMed Central

    Parasassi, T; De Stasio, G; Rusch, R M; Gratton, E

    1991-01-01

    The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles, there is an additional feature of the model, which is related to the apparent distribution of the rate R12. Significantly better fits were obtained using a continuous lorentzian distribution of interconversion rates. The resulting lifetime distribution was asymmetric and showed a definite narrowing above the phase transition. Images FIGURE 6 PMID:2009361

  16. Flip-flop of oleic acid in a phospholipid membrane: rate and mechanism.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2014-11-13

    Flip-flop of protonated oleic acid molecules dissolved at two different concentrations in membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is studied with the aid of molecular dynamics simulations at a time scale of several microseconds. Direct, single-molecule flip-flop events are observed at this time scale, and the flip-flop rate is estimated at 0.2-0.3 μs(-1). As oleic acid molecules move toward the center of the bilayer during flip-flop, they undergo gradual, correlated translational, and rotational motion. Rare, double-flipping events of two hydrogen-bonded oleic acid molecules are also observed. A two-dimensional free energy surface is obtained for the translational and rotational degree of freedom of the oleic acid molecule, and the minimum energy path on this surface is determined. A barrier to flip-flop of ~4.2 kcal/mol is found at the center of the bilayer. A two-dimensional diffusion model is found to provide a good description of the flip-flop process. The fast flip-flop rate lends support to the proposal that fatty acids permeate membranes without assistance of transport proteins. It also suggests that desorption rather than flip-flop is the rate-limiting step in fatty acid transport through membranes. The relation of flip-flop rates to the evolution of ancestral cellular systems is discussed. PMID:25319959

  17. Permeation of aldopentoses and nucleosides through fatty acid and phospholipid membranes: implications to the origins of life.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2013-02-01

    Permeation of aldopentoses and nucleosides through fatty acid and phospholipid membranes was investigated by way of molecular dynamics simulations. Calculated permeability coefficients of membranes to aldopentoses, which exist predominantly in the pyranose form, are in a very good agreement with experimental results. The unexpected preferential permeation of ribose, compared to its diastereomers, found by Sacerdote and Szostak, is explained in terms of inter- and intramolecular interactions involving hydroxyl groups. In aqueous solution, these groups favor the formation of intermolecular hydrogen bonds with neighboring water molecules. Inside the membrane, however, they form intramolecular hydrogen bonds, which in ribose are arranged in a chain. In its diastereomers this chain is broken, which yields higher free energy barrier to transfer through membranes. Faster permeation of ribose would lead to its preferential accumulation inside cells if sugars were converted sufficiently quickly to nonpermeable derivatives. An estimate for the rate of such reaction was derived. Preferential accumulation of ribose would increase the probability of correct monomers' incorporation during synthesis of nucleic acids inside protocells. The same mechanism does not apply to nucleosides or their activated derivatives because sugars are locked in the furanose form, which contains fewer exocyclic hydroxyl groups than does pyranose. The results of this study underscore concerted early evolution of membranes and the biochemical processes that they encapsulated. PMID:23397957

  18. How small polar molecules protect membrane systems against osmotic stress: the urea-water-phospholipid system.

    PubMed

    Costa-Balogh, Fátima O; Wennerström, Håkan; Wadsö, Lars; Sparr, Emma

    2006-11-30

    We investigate how a small polar molecule, urea, can act to protect a phospholipid bilayer system against osmotic stress. Osmotic stress can be caused by a dry environment, by freezing, or by exposure to aqueous systems with high osmotic pressure due to solutes like in saline water. A large number of organisms regularly experience osmotic stress, and it is a common response to produce small polar molecules intracellularly. We have selected a ternary system of urea-water-dimyristoyl phosphatidylcholine (DMPC) as a model to investigate the molecular mechanism behind this protective effect, in this case, of urea, and we put special emphasis on the applications of urea in skin care products. Using differential scanning calorimetry, X-ray diffraction, and sorption microbalance measurements, we studied the phase behavior of lipid systems exposed to an excess of solvent of varying compositions, as well as lipid systems exposed to water at reduced relative humidities. From this, we have arrived at a rather detailed thermodynamic characterization. The basic findings are as follows: (i) In excess solvent, the thermally induced lipid phase transitions are only marginally dependent on the urea content, with the exception being that the P(beta) phase is not observed in the presence of urea. (ii) For lipid systems with limited access to solvent, the phase behavior is basically determined by the amount (volume) of solvent irrespective of the urea content. (iii) The presence of urea has the effect of retaining the liquid crystalline phase at relative humidities down to 64% (at 27 degrees C), whereas, in the absence of urea, the transition to the gel phase occurs already at a relative humidity of 94%. This demonstrates the protective effect of urea against osmotic stress. (iv) In skin care products, urea is referred to as a moisturizer, which we find slightly misleading as it replaces the water while keeping the physical properties unaltered. (v) In other systems, urea is known to

  19. Ordered and disordered phospholipid domains coexist in membranes containing the calcium pump protein of sarcoplasmic reticulum.

    PubMed Central

    Lentz, B R; Clubb, K W; Barrow, D A; Meissner, G

    1983-01-01

    Data are presented that lead to an alternative model for the organization and molecular dynamics of lipid molecules near the Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) of sarcoplasmic reticulum. Measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in progressively delipidated sarcoplasmic reticulum membranes have been quantitatively interpreted in terms of a layer of lipid of high anisotropy (the lipid annulus) coexisting with lipid layers of very low anisotropy. In addition, the Ca2+-ATPase has been reconstituted into pure 1,2-dipentadecanoyl 3-sn-phosphatidylcholine membranes over a range of lipid-to-protein ratios. High-sensitivity differential scanning calorimetry has demonstrated that roughly 30 lipid molecules per Ca2+-ATPase molecule (annular lipids) fail to undergo a calorimetrically detectable phase transition in the temperature range 4-44 degrees C. Roughly 100 lipid molecules beyond the annulus undergo a detectable phase transition at a temperature below the phase transition of pure lipid and with an enthalpy change [4.2 kcal/mol (1 kcal = 4.18 kJ)] about half that observed for pure lipid vesicles (7.7-7.8 kcal/mol). We propose that both the fluorometric and calorimetric data are consistent with a model in which a motionally inhibited lipid annulus is surrounded by a more extensive region of disrupted lipid packing order, which we have called the secondary lipid domain. PMID:6222375

  20. Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa.

    PubMed

    Macías García, B; González Fernández, L; Ortega Ferrusola, C; Morillo Rodríguez, A; Gallardo Bolaños, J M; Rodríguez Martinez, H; Tapia, J A; Morcuende, D; Peña, F J

    2011-03-15

    Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32. PMID:21144567

  1. Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity.

    PubMed

    Tomek, Petr; Hrouzek, Pavel; Kuzma, Marek; Sýkora, Jan; Fiser, Radovan; Cerný, Jan; Novák, Petr; Bártová, Simona; Simek, Petr; Hof, Martin; Kavan, Daniel; Kopecký, Jirí

    2015-02-16

    There is mounting evidence that cyanobacterial lipopeptides can kill mammalian cells, presenting a hazard to human health. Unfortunately, their mechanism of toxicity is poorly understood. We have isolated new cyclic undecalipopeptides muscotoxin A and B containing unique lipophilicresidue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa). Muscotoxin B was not used for biological studies due to its poor yield. Muscotoxin A was cytotoxic to YAC-1, Sp/2, and HeLa cancer cell lines (LC(50) ranged from 9.9 to 13.2 μM after 24 h of exposure), causing membrane damage and influx of calcium ions. Subsequently, we studied this lytic mechanism using synthetic liposomes with encapsulated fluorescent probes. Muscotoxin A permeabilized liposomes composed exclusively of phospholipids, demonstrating that no proteins or carbohydrates present in biomembranes are essential for its activity. Paradoxically, the permeabilization activity of muscotoxin A was mediated by a significant reduction in membrane surface fluidity (stiffening), the opposite of that caused by synthetic detergents and cytolytic lipopeptide puwainaphycin F. At 25 °C, muscotoxin A disrupted liposomes with and without cholesterol/sphingomyelin; however, at 37 °C, it was selective against liposomes with cholesterol/sphingomyelin. It appears that both membrane fluidity and organization can affect the lytic activity of muscotoxin A. Our findings strengthen the evidence that cyanobacterial lipopeptides specifically disrupt mammalian cell membranes and bring new insights into the mechanism of this effect. PMID:25621379

  2. Aggregation of oligoarginines at phospholipid membranes: molecular dynamics simulations, time-dependent fluorescence shift, and biomimetic colorimetric assays.

    PubMed

    Vazdar, Mario; Wernersson, Erik; Khabiri, Morteza; Cwiklik, Lukasz; Jurkiewicz, Piotr; Hof, Martin; Mann, Ella; Kolusheva, Sofiya; Jelinek, Raz; Jungwirth, Pavel

    2013-10-01

    A time-dependent fluorescence shift method, biomimetic colorimetric assays, and molecular dynamics simulations have been performed in search of explanations why arginine rich peptides with intermediate lengths of about 10 amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. First, we demonstrate that an important factor for efficient peptide adsorption, as the first prerequisite for translocation across the membrane, is the presence of negatively charged phospholipids in the bilayer. Second, we observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface, which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides. PMID:24020922

  3. Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

    PubMed

    Podoplelova, Nadezhda A; Sveshnikova, Anastasia N; Kurasawa, James H; Sarafanov, Andrey G; Chambost, Herve; Vasil'ev, Sergey A; Demina, Irina A; Ataullakhanov, Fazly I; Alessi, Marie-Christine; Panteleev, Mikhail A

    2016-06-01

    Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow. PMID:26874201

  4. Hydration strongly affects the molecular and electronic structure of membrane phospholipids

    NASA Astrophysics Data System (ADS)

    Mashaghi, Alireza; Partovi-Azar, P.; Jadidi, Tayebeh; Nafari, Nasser; Maass, Philipp; Tabar, M. Reza Rahimi; Bonn, Mischa; Bakker, Huib J.

    2012-03-01

    We investigate the structure and electronic properties of phosphatidylcholine (PC) under different degrees of hydration at the single-molecule and monolayer type level by linear scaling ab initio calculations. Upon hydration, the phospholipid undergoes drastic long-range conformational rearrangements which lead to a sickle-like ground-state shape. The structural unit of the tilted gel-phase PC appears to be a water-bridged PC dimer. We find that hydration dramatically alters the surface potential, dipole and quadrupole moments of the lipids and consequently guides the interactions of the lipids with other molecules and the communication between cells.

  5. The effects of chondroitin sulfate and serum albumin on the fibrillation of human islet amyloid polypeptide at phospholipid membranes.

    PubMed

    Li, Yang; Wang, Li; Lu, Tong; Wei, Ying; Li, Fei

    2016-04-28

    Glycosaminoglycans and serum albumin are important cellular components that regulate the fibril formation of proteins. Whereas the effects of cellular components on the fibrillation of amyloid proteins in bulk solution are widely studied, less attention has been paid to the effects of cellular components on amyloidogenesis occurring at cellular membranes. In this study, we focus on the impacts of chondroitin sulfate A (CSA) and bovine serum albumin (BSA) on the amyloidogenic behaviors of human islet amyloid polypeptide (hIAPP) at phospholipid membranes consisting of neutral POPC and anionic POPG. Using the thioflavin T fluorescence assay, atomic force microscopy, circular dichroism and nuclear magnetic resonance measurements, we demonstrate that CSA has an intensive promotion effect on the fibrillation of hIAPP at the POPC membrane, which is larger than the total effect of CSA alone and POPC alone. The further enhanced promotion of the fibrillation of hIAPP by CSA at the neutral membrane is associated with a specific interaction of CSA with POPC. In contrast, the activity of BSA as an inhibitor of hIAPP fibrillation observed in bulk solution decreases dramatically in the presence of POPG vesicles. The dramatic loss of the inhibition efficiency of BSA arises essentially from a specific interaction with the POPG component, but not simply from suppression by an opposite effect of the anionic membrane. The findings in this study suggest that the interactions between membranes and cellular components may have a significant effect on the activity of the cellular components in regulating the fibrillation of hIAPP. PMID:27067251

  6. The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: a physico-chemical study with lecithin phospholipids.

    PubMed

    Manrique-Moreno, Marcela; Moreno, Marcela Manrique; Garidel, Patrick; Suwalsky, Mario; Howe, Jörg; Brandenburg, Klaus

    2009-06-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain-chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T(m) a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface. PMID:19366589

  7. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    PubMed

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription. PMID:24960695

  8. Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria

    PubMed Central

    Bishop, Russell E.; Gibbons, Henry S.; Guina, Tina; Trent, M.Stephen; Miller, Samuel I.; Raetz, Christian R.H.

    2000-01-01

    Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His6-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg2+ limitation and host innate immune defenses. PMID:11013210

  9. Etoposide incorporated into camel milk phospholipids liposomes shows increased activity against fibrosarcoma in a mouse model.

    PubMed

    Maswadeh, Hamzah M; Aljarbou, Ahmad N; Alorainy, Mohammed S; Alsharidah, Mansour S; Khan, Masood A

    2015-01-01

    Phospholipids were isolated from camel milk and identified by using high performance liquid chromatography and gas chromatography-mass spectrometry (GC/MS). Anticancer drug etoposide (ETP) was entrapped in liposomes, prepared from camel milk phospholipids, to determine its activity against fibrosarcoma in a murine model. Fibrosarcoma was induced in mice by injecting benzopyrene (BAP) and tumor-bearing mice were treated with various formulations of etoposide, including etoposide entrapped camel milk phospholipids liposomes (ETP-Cam-liposomes) and etoposide-loaded DPPC-liposomes (ETP-DPPC-liposomes). The tumor-bearing mice treated with ETP-Cam-liposomes showed slow progression of tumors and increased survival compared to free ETP or ETP-DPPC-liposomes. These results suggest that ETP-Cam-liposomes may prove to be a better drug delivery system for anticancer drugs. PMID:25821817

  10. Omega-3 DHA and EPA for cognition, behavior, and mood: clinical findings and structural-functional synergies with cell membrane phospholipids.

    PubMed

    Kidd, Parris M

    2007-09-01

    The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are orthomolecular, conditionally essential nutrients that enhance quality of life and lower the risk of premature death. They function exclusively via cell membranes, in which they are anchored by phospholipid molecules. DHA is proven essential to pre- and postnatal brain development, whereas EPA seems more influential on behavior and mood. Both DHA and EPA generate neuroprotective metabolites. In double-blind, randomized, controlled trials, DHA and EPA combinations have been shown to benefit attention deficit/hyperactivity disorder (AD/HD), autism, dyspraxia, dyslexia, and aggression. For the affective disorders, meta-analyses confirm benefits in major depressive disorder (MDD) and bipolar disorder, with promising results in schizophrenia and initial benefit for borderline personality disorder. Accelerated cognitive decline and mild cognitive impairment (MCI) correlate with lowered tissue levels of DHA/EPA, and supplementation has improved cognitive function. Huntington disease has responded to EPA. Omega-3 phospholipid supplements that combine DHA/EPA and phospholipids into the same molecule have shown marked promise in early clinical trials. Phosphatidylserine with DHA/EPA attached (Omega-3 PS) has been shown to alleviate AD/HD symptoms. Krill omega-3 phospholipids, containing mostly phosphatidylcholine (PC) with DHA/EPA attached, markedly outperformed conventional fish oil DHA/EPA triglycerides in double-blind trials for premenstrual syndrome/dysmenorrhea and for normalizing blood lipid profiles. Krill omega-3 phospholipids demonstrated anti-inflammatory activity, lowering C-reactive protein (CRP) levels in a double-blind trial. Utilizing DHA and EPA together with phospholipids and membrane antioxidants to achieve a triple cell membrane synergy may further diversify their currently wide range of clinical applications. PMID:18072818

  11. Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles.

    PubMed

    Wheaten, Sterling A; Ablan, Francis D O; Spaller, B Logan; Trieu, Julie M; Almeida, Paulo F

    2013-11-01

    The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. However, if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energies of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period, but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 min. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. However, some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle. PMID:24152283

  12. Ultrasonic study of melittin effects on phospholipid model membranes.

    PubMed Central

    Colotto, A; Kharakoz, D P; Lohner, K; Laggner, P

    1993-01-01

    Low dose effects of melittin on dilute suspensions of dipalmitoylphosphatidylcholine multilamellar vesicles are investigated by studying the acoustic properties of the system. The temperature dependencies of sound velocity and absorption have been measured at 7.2 MHz in the temperature range of 20-55 degrees C, for different peptide/lipid molar ratios, R. The most pronounced effects were observed at R = 5 x 10(-3), in the vicinity of the pretransition, with a simultaneous increase in sound absorption and velocity. This indicates that melittin affects the polar head group region of the bilayer resulting in a decrease in mobility of the polar head groups. A nonmonotonic dependence of the main transition temperature, with an initial decrease followed by an increase as melittin is added, is interpreted as a consequence of a destabilizing action of the interfaces between mellitin-affected clusters and the unaffected phase. PMID:8312475

  13. Inward multivesiculation at the basal membrane of adherent giant phospholipid vesicles.

    PubMed

    Moreno-Flores, Susana

    2016-04-01

    Adherent giant vesicles composed of phosphatidylcholine, phosphatidylserine and biotinylated lipids form clusters of inward spherical buds at their basal membrane. The process is spontaneous and occurs when the vesicles undergo a sequence of osmotic swelling and deswelling. The daughter vesicles have a uniform size (diameter ≈ 2-3 μm), engulf small volumes of outer fluid and remain attached to the region of the membrane from which they generate, even after restoring the isotonicity. A pinning-sealing mechanism of long-wavelength modes of membrane fluctuations is proposed, by which the just-deflated vesicles reduce the surplus of membrane area and avoid excessive spreading and compression via biotin anchors. The work discusses the rationale behind the mechanism that furnishes GUVs with basal endovesicles, and its prospective use to simulate cellular events or to create molecular carriers. PMID:26828120

  14. Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids.

    PubMed Central

    Mynatt, R L; Greenhaw, J J; Cook, G A

    1994-01-01

    It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I. PMID:8192665

  15. Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus

    SciTech Connect

    de Bony, J.; Lopez, A.; Gilleron, M.; Welby, M.; Laneelle, G.; Rousseau, B.; Beaucourt, J.P.; Tocanne, J.F. )

    1989-05-02

    The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

  16. Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins.

    PubMed

    Larsen, Andreas N; Sørensen, Kasper K; Johansen, Nicolai T; Martel, Anne; Kirkensgaard, Jacob J K; Jensen, Knud J; Arleth, Lise; Midtgaard, Søren Roi

    2016-07-01

    Three dimers of the amphipathic α-helical peptide 18A have been synthesized with different interhelical linkers inserted between the two copies of 18A. The dimeric peptides were denoted 'beltides' where Beltide-1 refers to the 18A-dimer without a linker, Beltide-2 is the 18A-dimer with proline (Pro) as a linker and Beltide-3 is the 18A-dimer linked by two glycines (Gly-Gly). The self-assembly of the beltides with the phospholipid DMPC was studied with and without the incorporated membrane protein bacteriorhodopsin (bR) through a combination of coarse-grained MD simulations, size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, small-angle scattering (SAS), static light scattering (SLS) and UV-Vis spectroscopy. For all three beltides, MD and combined small-angle X-ray and -neutron scattering were consistent with a disc structure composed by a phospholipid bilayer surrounded by a belt of peptides and with a total disc diameter of approximately 10 nm. CD confirmed that all three beltides were α-helical in the free form and with DMPC. However, as shown by SEC the different interhelical linkers clearly led to different properties of the beltides. Beltide-3, with the Gly-Gly linker, was very adaptable such that peptide nanodiscs could be formed for a broad range of different peptide to lipid stoichiometries and therefore also possible disc-sizes. On the other hand, both Beltide-2 with the Pro linker and Beltide-1 without a linker were less adaptable and would only form discs of certain peptide to lipid stoichiometries. SLS revealed that the structural stability of the formed peptide nanodiscs was also highly affected by the linkers and it was found that Beltide-1 gave more stable discs than the other two beltides. With respect to membrane protein stabilization, each of the three beltides in combination with DMPC stabilizes the seven-helix transmembrane protein bacteriorhodopsin significantly better than the detergent octyl glucoside, but no

  17. Retardation of Abeta fibril formation by phospholipid vesicles depends on membrane phase behavior.

    PubMed

    Hellstrand, Erik; Sparr, Emma; Linse, Sara

    2010-05-19

    An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Abeta). Recombinant Abeta(M1-40) and Abeta(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Abeta fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane. PMID:20483329

  18. Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

    PubMed Central

    Sprícigo, José F. W.; Diógenes, Mateus N.; Leme, Ligiane O.; Guimarães, Ana L.; Muterlle, Carolle V.; Silva, Bianca Damiani Marques; Solà-Oriol, David; Pivato, Ivo; Silva, Luciano Paulino; Dode, Margot A. N.

    2015-01-01

    The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was

  19. Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes.

    PubMed

    Antonenko, Yuri N; Perevoshchikova, Irina V; Davydova, Lyubov I; Agapov, Igor A; Bogush, Vladimir G

    2010-06-01

    Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core. PMID:20214876

  20. Binding of the Three-Repeat Domain of Tau to Phospholipid Membranes induces an Aggregated-Like State of the Protein

    PubMed Central

    Künze, Georg; Barré, Patrick; Scheidt, Holger A.; Thomas, Lars; Eliezer, David; Huster, Daniel

    2013-01-01

    In patients with Alzheimer’s disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H-13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau. PMID:22521809

  1. Interactions of Amelogenin with Phospholipids

    PubMed Central

    Lokappa, Sowmya Bekshe; Chandrababu, Karthik Balakrishna; Dutta, Kaushik; Perovic, Iva; Evans, John Spencer; Moradian-Oldak, Janet

    2015-01-01

    Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, towards the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (~334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. Though in DLS studies we cannot exclude the possibility of fusion of liposomes as the result of amelogenin addition, NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong FRET from Trp in rP172 to DNS–bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. PMID:25298002

  2. On the application of the MARTINI coarse-grained model to immersion of a protein in a phospholipid bilayer

    NASA Astrophysics Data System (ADS)

    Mustafa, Ghulam; Nandekar, Prajwal P.; Yu, Xiaofeng; Wade, Rebecca C.

    2015-12-01

    An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions.

  3. On the application of the MARTINI coarse-grained model to immersion of a protein in a phospholipid bilayer

    SciTech Connect

    Mustafa, Ghulam E-mail: rebecca.wade@h-its.org; Nandekar, Prajwal P.; Yu, Xiaofeng; Wade, Rebecca C. E-mail: rebecca.wade@h-its.org

    2015-12-28

    An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions.

  4. Cell signalling and phospholipid metabolism

    SciTech Connect

    Boss, W.F.

    1989-01-01

    Our research for the past two years has involved the study of phosphoinositides and their potential role in regulating plant growth and development. Our initial goal was to document the sequence of events involved in inositol phospholipid metabolism in response to external stimuli. Our working hypothesis was that phosphatidylinositol bisphosphate (PIP/sub 2/) was in the plasma membrane of plants cells and would be hydrolyzed by phospholipase C to yield the second messengers inositol triphosphate (IP/sub 3/) and diacyglycerol (DAG) and that IP/sub 3/ would mobilize intracellular calcium as has been shown for animal cells. Our results with both carrot suspension culture cells and sunflower hypocotyl indicate that this paradigm is not the primary mechanism of signal transduction in these systems. We have observed very rapid, within 5 sec, stimulation of phosphatidylinositol monophosphate (PIP) kinase which resulted in an increase in PIP/sub 2/. However, there was no evidence for activation of phospholipase C. In addition, we have shown that PIP and PIP/sub 2/ can activate the plasma membrane ATPase. The results of these studies are described briefly in the paragraphs below. Inositol phospholipids are localized in distinct membrane fractions. If PIP and PIP/sub 2/ play a role in the transduction of external signals, they should be present in the plasma membrane. We used the fusogenic carrot suspension culture cells as a model system to study the distribution of inositol phospholipids in various membrane fractions and organelles. Cells were labeled 12 to 18 h with myo(2-/sup 3/H) inositol and the membranes were isolated by aqueous two-phase partitioning. The plasma membrane was enriched in PIP and PIP/sub 2/ compared to the intracellular membranes.

  5. Ubiquinol and plastoquinol triphenylphosphonium conjugates can carry electrons through phospholipid membranes.

    PubMed

    Rokitskaya, Tatyana I; Murphy, Michael P; Skulachev, Vladimir P; Antonenko, Yuri N

    2016-10-01

    Many mitochondria-targeted antioxidants (MTAs) that comprise a quinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation are widely used for evaluating the role of mitochondria in pathological processes involving oxidative stress. The potency of MTAs to carry electrons across biological membranes and thereby mediate transmembrane redox processes was unknown. To assess this, we measured the rate of ferricyanide reduction inside liposomes by external ascorbate. Here, we show that MTAs containing ubiquinone (MitoQ series) or plastoquinone (SkQ series) can carry electrons through lipid membranes, with the rate being inversely proportional to the length of the hydrocarbon linker group. Furthermore, this process was stimulated by the hydrophobic anion tetraphenylborate suggesting that permeation of the cationic MTA through the membrane was the rate-limiting step of the process. This conclusion was supported by the observation that the rate of MTA-induced electron transfer was insensitive to nigericin, in contrast to electron transfer mediated by neutral quinone derivatives. These findings indicate that MTAs can be utilized to transfer electrons across lipid membranes and this may be applicable to the study of the electron-transport chain in mitochondria and other natural membranes exhibiting redox processes. PMID:27182824

  6. MICOS and phospholipid transfer by Ups2-Mdm35 organize membrane lipid synthesis in mitochondria.

    PubMed

    Aaltonen, Mari J; Friedman, Jonathan R; Osman, Christof; Salin, Bénédicte; di Rago, Jean-Paul; Nunnari, Jodi; Langer, Thomas; Tatsuta, Takashi

    2016-06-01

    Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function. PMID:27241913

  7. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  8. Bending membranes on demand: fluid phospholipid bilayers on topographically deformable substrates.

    PubMed

    Sanii, Babak; Smith, Andreia Michelle; Butti, Ravichandra; Brozell, Adrian M; Parikh, Atul N

    2008-03-01

    We combine hierarchical surface wrinkling of elastomers with lipid membrane deposition techniques to dynamically template complex three-dimensional topographies onto supported lipid bilayers. The real-time introduction of corresponding nano- to micrometer scale curvatures triggers spatially periodic, elastic bending of the bilayer, accompanied by molecular-level reorganizations. This ability to dynamically impose curvatures on supported bilayers and the ensuing re-equilibration promises fundamental material and biophysical investigations of curvature-dependent, static heterogeneities and dynamic reorganizations pervasive in biological membranes. PMID:18271562

  9. Interaction of melittin with mixed phospholipid membranes composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine studied by deuterium NMR

    SciTech Connect

    Dempsey, C.; Bitbol, M.; Watts, A. )

    1989-08-08

    The interaction of bee venom melittin with mixed phospholipid bilayers composed of dimyristoylphosphatidylcholine deuterated in the {alpha}- and {beta}-methylenes of the choline head group (DMPC-d{sub 4}) and dimyristoylphosphatidylserine deuterated in the {alpha}-methylene and {beta}-CH positions of the serine head group (DMPS-d{sub 3}) was studied in ternary mixtures by using deuterium NMR spectroscopy. The changes in the deuterium quadrupole splittings of the head-group deuteriomethylenes of DMPC-d{sub 4} induced by DMPS in binary mixtures were systematically reversed by increasing concentrations of melittin, so that at a melittin concentration of 4 mol % relative to total lipid the deuterium NMR spectrum from DMPC-d{sub 4} in the ternary mixture was similar to the spectrum from pure DMPC-d{sub 4} bilayers. The absence of deuterium NMR signals arising from melittin-bound DMPS in ternary mixtures containing DMPS-d{sub 3} indicates that the reversal by melittin of the effects of DMPS on the quadrupole splittings of DMPC-d{sub 4} results from the response of the choline head group to the net surface charge rather than from phase separation of melittin-DMPS complexes. The similarity in the effects of the two cationic but otherwise dissimilar peptides indicates that the DMPS head group responds to the surface charge resulting from the presence in the bilayer of charged amphiphiles, in a manner analogous to the response of the choline head group of phosphatidylcholine to the bilayer surface charge. The presence of DMPS greatly stabilized DMPC bilayers with respect to melittin-induced micellization, indicating that the latter effect of melittin may not be important for the hemolytic activity of the peptide.

  10. Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.

    PubMed

    Jablonická, Veronika; Mansfeld, Johanna; Heilmann, Ingo; Obložinský, Marek; Heilmann, Mareike

    2016-09-01

    The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. PsPLA2 contains a PLA2 signature domain (PA2c), including the Ca(2+)-binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the PsPLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature PsPLA2 is 14.0 kDa. Based on the primary structure PsPLA2 belongs to the XIB group of PLA2s. Untagged recombinant PsPLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro. The pH optimum for activity of PsPLA2 was found to be pH 7, when using mixed micelles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Triton X-100. PsPLA2 specifically cleaves fatty acids from the sn-2 position of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids. PMID:27473012

  11. Feruloyl Dioleoyglycerol Antioxidant Capacity in Phospholipid Vesicles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferulic acid and its esters are known to be effective antioxidants. Feruloyl dioleoylglycerol was assessed for its ability to serve as an antioxidant in model membrane phospholipid vesicles. The molecule was incorporated into single-lamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine at ...

  12. Ion transport through chemically induced pores in protein-free phospholipid membranes.

    PubMed

    Gurtovenko, Andrey A; Anwar, Jamshed

    2007-11-29

    We address the possibility of being able to induce the trafficking of salt ions and other solutes across cell membranes without the use of specific protein-based transporters or pumps. On the basis of realistic atomic-scale molecular dynamics simulations, we demonstrate that transmembrane ionic leakage can be initiated by chemical means, in this instance through addition of dimethyl sulfoxide (DMSO), a solvent widely used in cell biology. Our results provide compelling evidence that the small amphiphilic solute DMSO is able to induce transient defects (water pores) in membranes and to promote a subsequent diffusive pore-mediated transport of salt ions. The findings are consistent with available experimental data and offer a molecular-level explanation for the experimentally observed activities of DMSO solvent as an efficient penetration enhancer and a cryoprotectant, as well as an analgesic. Our findings suggest that transient pore formation by chemical means could emerge as an important general principle for therapeutics. PMID:17983219

  13. Interactions of borneol with DPPC phospholipid membranes: a molecular dynamics simulation study.

    PubMed

    Yin, Qianqian; Shi, Xinyuan; Ding, Haiou; Dai, Xingxing; Wan, Guang; Qiao, Yanjiang

    2014-01-01

    Borneol, known as a "guide" drug in traditional Chinese medicine, is widely used as a natural penetration enhancer in modern clinical applications. Despite a large number of experimental studies on borneol's penetration enhancing effect, the molecular basis of its action on bio-membranes is still unclear. We carried out a series of coarse-grained molecular dynamics simulations with the borneol concentration ranging from 3.31% to 54.59% (v/v, lipid-free basis) to study the interactions of borneol with aDPPC(1,2-dipalmitoylsn-glycero-3-phosphatidylcholine) bilayer membrane, and the temperature effects were also considered. At concentrations below 21.89%, borneol's presence only caused DPPC bilayer thinning and an increase in fluidity; A rise in temperature could promote the diffusing progress of borneol. When the concentration was 21.89% or above, inverted micelle-like structures were formed within the bilayer interior, which led to increased bilayer thickness, and an optimum temperature was found for the interaction of borneol with the DPPC bilayer membrane. These findings revealed that the choice of optimal concentration and temperature is critical for a given application in which borneol is used as a penetration enhancer. Our results not only clarify some molecular basis for borneol's penetration enhancing effects, but also provide some guidance for the development and applications of new preparations containing borneol. PMID:25383679

  14. Interaction between active ruthenium complex [RuCl3(dppb)(VPy)] and phospholipid Langmuir monolayers: Effects on membrane electrical properties

    NASA Astrophysics Data System (ADS)

    Sandrino, B.; Wrobel, E. C.; Nobre, T. M.; Caseli, L.; Lazaro, S. R.; Júnior, A. C.; Garcia, J. R.; Oliveira, O. N.; Wohnrath, K.

    2016-04-01

    We report on the interaction between mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis(diphenylphosphine)butane and VPy = 4-vinylpyridine) (RuVPy) and dipalmitoyl phosphatidyl serine (DPPS), in Langmuir and Langmuir-Blodgett (LB) films. Interaction of RuVPy with DPPS, which predominates in cancer cell membranes, should be weaker than for other phospholipids since RuVPy is less toxic to cancer cells than to healthy cells. Incorporation of RuVPy induced smaller changes in electrochemical properties of LB films of DPPS than for other phospholipids, but the same did not apply to surface pressure isotherms. This calls for caution in establishing correlations between effects from a single property and phenomena on cell membranes.

  15. Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

    PubMed Central

    Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

    2016-01-01

    Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

  16. Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

    PubMed

    Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

    2016-01-01

    Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

  17. General model of phospholipid bilayers in fluid phase within the single chain mean field theory

    SciTech Connect

    Guo, Yachong; Baulin, Vladimir A.; Pogodin, Sergey

    2014-05-07

    Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

  18. Lipid metabolizing enzyme activities modulated by phospholipid substrate lateral distribution.

    PubMed

    Salinas, Dino G; Reyes, Juan G; De la Fuente, Milton

    2011-09-01

    Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters--without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes. PMID:21108012

  19. Response of Halomonas campisalis to saline stress: changes in growth kinetics, compatible solute production and membrane phospholipid fatty acid composition.

    PubMed

    Aston, John E; Peyton, Brent M

    2007-09-01

    The haloalkaliphile Halomonas campisalis, isolated near Soap Lake, Washington, was grown under both aerobic and denitrifying conditions from 0 to 260 g L(-1) NaCl, with optimal growth occurring at 20 and 30 g L(-1) NaCl, respectively. Halomonas campisalis was observed to produce high concentrations of compatible solutes, most notably ectoine (up to 500 mM within the cytoplasm), but hydroxyectoine and glycine betaine were also detected. The types and amounts of compatible solutes produced depended on salinity and specific growth rate, as well as on the terminal electron acceptor available (O(2) or NO(3) (-)). A decrease in ectoine production was observed with NO(3) (-) as compared with O(2) as the terminal electron acceptor. In addition, changes in the phospholipid fatty acid composition were measured with changing salinity. An increase in trans fatty acids was observed in the absence of salinity, and may be a response to membrane instability. Cyclic fatty acids were also observed to increase, both in the absence of salinity, and at very high salinities, indicating cell stress at these conditions. PMID:17651393

  20. Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

    PubMed Central

    Fernandes, K.S.; Silva, A.H.M.; Mendanha, S.A.; Rezende, K.R.; Alonso, A.

    2013-01-01

    4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation. PMID:24068194

  1. Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli.

    PubMed Central

    Hardaway, K L; Buller, C S

    1979-01-01

    Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A. PMID:104974

  2. Acyl chain length effects related to glycosphingolipid crypticity in phospholipid membranes: probed by 2H-NMR.

    PubMed

    Hamilton, K S; Briere, K; Jarrell, H C; Grant, C W

    1994-03-23

    Wideline 2H-NMR was used to consider the relationships amongst glycosphingolipid and phospholipid fatty acid chain length and glycosphingolipid receptor function, in a system classically associated with crypticity. Galactosyl ceramide (GalCer), having 18- or 24-carbon fatty acid, was deuterium labelled at the conformationally-restricted fatty acid alpha-carbon (C-2). 2H-NMR spectra of N-[2,2-2H2]stearoyl and N-[2,2-2H2]lignoceroyl GalCer (GalCer with 18-vs. 24-carbon selectively deuterated fatty acid) were then compared over a range of temperatures in phosphatidylcholine/cholesterol membranes in which the host phospholipid had dimyristoyl, dipalmitoyl, or distearoyl fatty acid composition. Findings were evaluated in the light of known sensitivity of antibody interaction with GalCer to temperature and to both glycolipid fatty acid chain length and host matrix fatty acid chain length. Under the conditions of experimentation, spectra were not obtainable for glycolipids having rigid body motions that were slow on the NMR timescale (10(-4)-10(-5) s)-i.e.. motions typical of non-fluid (gel phase) membranes. The systems, DPPC/cholesterol and DSPC/cholesterol, in which the original observation was made of increased antibody binding to GalCer with long fatty acid, proved to be characterised by receptor motions that were in this slow timescale for both 18:0 and 24:0 GalCer at 22-24 degrees C. Under conditions for which spectra could be obtained, those for GalCer with [2,2-2H2]lignoceroyl (24-carbon alpha-deuterated) fatty acid were qualitatively similar to those of its 18-carbon analogue in all (fluid) membranes examined. However, spectral splittings differed quantitatively between deuterated 18:0 and 24:0 GalCer at a given temperature, dependent upon host matrix. These differences were most marked at lower temperatures and in the longer chain (more ordered) matrices, DPPC/cholesterol and DSPC/cholesterol. This suggests that maximum effects of glycolipid chain length on

  3. Infrared spectra of phospholipid membranes: interfacial dehydration by volatile anesthetics and phase transition.

    PubMed

    Tsai, Y S; Ma, S M; Nishimura, S; Ueda, I

    1990-02-28

    Fourier-transform infrared attenuated total reflection (ATR) spectroscopy was used to study the effect of volatile anesthetics on fully hydrated dipalmitoylphosphatidylcholine (DPPC) vesicle membranes. The main phase transition was monitored by the change in the C-H2 asymmetric stretching frequencies of the lipid tails. The surface property was analyzed by the changes in the P = O stretching, (CH3)3-N+ stretching of the hydrophilic head, and C = O stretching of the glycerol skeleton. The partial pressures of those agents that decreased the transition temperature 1.0 C degree were halothane 0.75, enflurane 1.90 and CCl4 0.85 kPa. At a 2:1 lipid/anesthetic mole ratio, the polar anesthetics, halothane and enflurane, increased the ratio of (P = O stretching band area)/((CH3)3-N+ stretching band area) by 26.3% and 21.1%, respectively, whereas apolar CCl4 increased it 10.5%. The water molecules bound to the P = O moiety are apparently replaced by the anesthetic molecules. The deconvoluted C = O spectra showed two peaks: free sn-1 that is closer to the lipid core and hydrogen-bonded sn-2 that is closer to the polar head. Addition of halothane and enflurane, but not CCl4, increased the number of peaks to three. The third peak is free sn-2, formed by disrupting hydrogen-bonding to water. Because the temperature-induced spectral change was limited to C-H2 stretching at the main phase transition, the effects of anesthetics on the lipid membrane structure are not identical to temperature elevation. Among anesthetics, the effects of apolar and polar molecules on the interfacial properties are different. PMID:2306457

  4. Higher sterol content regulated by CYP51 with concomitant lower phospholipid content in membranes is a common strategy for aluminium tolerance in several plant species.

    PubMed

    Wagatsuma, Tadao; Khan, Md Shahadat Hossain; Watanabe, Toshihiro; Maejima, Eriko; Sekimoto, Hitoshi; Yokota, Takao; Nakano, Takeshi; Toyomasu, Tomonobu; Tawaraya, Keitaro; Koyama, Hiroyuki; Uemura, Matsuo; Ishikawa, Satoru; Ikka, Takashi; Ishikawa, Akifumi; Kawamura, Takeshi; Murakami, Satoshi; Ueki, Nozomi; Umetsu, Asami; Kannari, Takayuki

    2015-02-01

    Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species. PMID:25416794

  5. Induction of intranuclear membranes by overproduction of Opi1p and Scs2p, regulators for yeast phospholipid biosynthesis, suggests a mechanism for Opi1p nuclear translocation.

    PubMed

    Masuda, Miki; Oshima, Ayaka; Noguchi, Tetsuko; Kagiwada, Satoshi

    2016-03-01

    In the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes is suppressed by the Opi1p negative regulator. Opi1p enters into the nucleoplasm from the nuclear membrane to suppress the gene expression under repressing conditions. The binding of Opi1p to the nuclear membrane requires an integral membrane protein, Scs2p and phosphatidic acid (PA). Although it is demonstrated that the association of Opi1p with membranes is affected by PA levels, how Opi1p dissociates from Scs2p is unknown. Here, we found that fluorescently labelled Opi1p accumulated on a perinuclear region in an Scs2p-dependent manner. Electron microscopic analyses indicated that the perinuclear region consists of intranuclear membranes, which may be formed by the invagination of the nuclear membrane due to the accumulation of Opi1p and Scs2p in a restricted area. As expected, localization of Opi1p and Scs2p in the intranuclear membranes was detected by immunoelectron microscopy. Biochemical analysis showed that Opi1p recovered in the membrane fraction was detergent insoluble while Scs2p was soluble, implying that Opi1p behaves differently from Scs2p in the fraction. We hypothesize that Opi1p dissociates from Scs2p after targeting to the nuclear membrane, making it possible to be released from the membrane quickly when PA levels decrease. PMID:26590299

  6. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.

    PubMed

    Andrade, Débora M; Clausen, Mathias P; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E; Hell, Stefan W; Lagerholm, B Christoffer; Eggeling, Christian

    2015-01-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. PMID:26118385

  7. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane - a minimally invasive investigation by STED-FCS

    NASA Astrophysics Data System (ADS)

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-06-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

  8. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo. PMID:1737779

  9. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

    PubMed Central

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-01-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. PMID:26118385

  10. Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes.

    PubMed

    Kono, K; Henmi, A; Takagishi, T

    1999-09-21

    To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes. PMID:10561483

  11. 250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes.

    PubMed Central

    Earle, K. A.; Moscicki, J. K.; Ge, M.; Budil, D. E.; Freed, J. H.

    1994-01-01

    Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing. The addition of GA to DPPC essentially suppressed the broad background signal observed in pure DPPC dispersions. PMID:7518705

  12. Modulating alignment of membrane proteins in liquid-crystalline and oriented gel media by changing the size and charge of phospholipid bicelles

    PubMed Central

    Lorieau, Justin L; Maltsev, Alexander S.; Louis, John M; Bax, Ad

    2013-01-01

    We demonstrate that alignment of a structured peptide or small protein solubilized in mixed phospholipid:detergent micelles or bicelles, when embedded in a compressed gel or liquid crystalline medium, can be altered by either changing the phospholipid aggregate shape, charge, or both together. For the hemagglutinin fusion peptide solubilized in bicelles, we show that bicelle shape and charge do not change its helical hairpin structure but impact its alignment relative to the alignment medium, both in charged compressed acrylamide gel and in liquid crystalline d (GpG). The method can be used to generate sets of residual dipolar couplings (RDCs) that correspond to orthogonal alignment tensors, and holds promise for high-resolution structural refinement and dynamic mapping of membrane proteins. PMID:23508769

  13. Adsorption of apolipoprotein A-I to biological membranes. A statistical mechanical model

    NASA Astrophysics Data System (ADS)

    Gross, Eitan

    2012-07-01

    Apolipoprotein A-I (apo A-I), the main protein component of high-density lipoprotein (HDL), reduces the risk for atherosclerosis by removing cholesterol from the membrane of foam cells. Experiments with model membrane systems have indicated, however, that membrane cholesterol reduces apo A-I binding to the membrane. Foam cells resolve this discrepancy electrostatically by co-inserting negatively charged phospholipids in their membrane. Here we present a statistical mechanical model to account for the effect of cholesterol. Our model is based on the Haugen and May model which takes into account the dipolar nature of the zwitterionic phospholipid head group in the membrane, in which the positive end of the zwitterionic dipole moment can move randomly on a hemispherical surface with a radius equal to the arm of the dipole moment and with the negative end fixed at the hydrocarbon layer. Adsorption of a positively charged apo A-I macroion to the surface of the membrane modifies the electric field within the head group region and induces lateral demixing of phospholipid molecules in the membrane. Results from numerical integration of model equations show that i) as a result of the strong charge-dipole electrostatic coupling, the positive end of the dipoles tilts away from the adsorbed macroion in a cooperative manner; and ii) cholesterol reduces macroion adsorption to the membrane by reducing the surface area of the membrane and restricting the dipoles range of rotation. Model predictions for the change in free energy of adsorption to zwitterionic membrane are in good agreement with previously reported experimental data with liposomes. The model can assist in designing new mimetic peptides.

  14. Formation and dynamics of supported phospholipid membranes on a periodic nanotextured substrate

    SciTech Connect

    Werner, James H; Montano, Gabriel M; Shreve, Andrew P

    2008-01-01

    We have studied and modeled the morphology and dynamics of planar lipid bilayer assemblies supported on a textured silicon substrate that contains nano-structured features perpendicular to the plane of observation. Using a combination of characterization methods, including atomic force microscopy and quantitative fluorescence microscopy, we have discovered the bilayer assemblies are conformal to the underlying nanostructured substrate. As far as the dynamics are concnerned, the lipids freely diffuse in and out of the plane of observation while supported by the nanotextured material. In a microscopic sense, diffusion is isotropic along the patterned substrate. Macroscopically, when observed over length scales exceeding the dimensions of the nano-structured features, the diffusion appears anisotropic. The observed macroscopic anisotropy is well simulated using models of diffusion on the nanostructured surface that assume the lipids diffuse homogeneously and isotropically on the supporting nanotextured substrate.

  15. Changes in the Fatty Acid Profile and Phospholipid Molecular Species Composition of Human Erythrocyte Membranes after Hybrid Palm and Extra Virgin Olive Oil Supplementation.

    PubMed

    Pacetti, D; Gagliardi, R; Balzano, M; Frega, N G; Ojeda, M L; Borrero, M; Ruiz, A; Lucci, P

    2016-07-13

    This work aims to evaluate and compare, for the first time, the effects of extra virgin olive oil (EVOO) and hybrid palm oil (HPO) supplementation on the fatty acid profile and phospholipid (PL) molecular species composition of human erythrocyte membranes. Results supported the effectiveness of both HPO and EVOO supplementation (3 months, 25 mL/day) in decreasing the lipophilic index of erythrocytes with no significant differences between HPO and EVOO groups at month 3. On the other hand, the novel and rapid ultraperformance liquid chromatography-tandem mass spectrometry method used for PL analysis reveals an increase in the levels of phosphatidylcholine and phosphatidylethanolamine species esterified with polyunsaturated fatty acids. This work demonstrates the ability of both EVOO and HPO to increase the degree of unsaturation of erythrocyte membrane lipids with an improvement in membrane fluidity that could be associated with a lower risk of developing cardiovascular diseases. PMID:27315139

  16. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness

    NASA Technical Reports Server (NTRS)

    Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W.

    1996-01-01

    Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.

  17. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness.

    PubMed Central

    Paula, S; Volkov, A G; Van Hoek, A N; Haines, T H; Deamer, D W

    1996-01-01

    Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths. PMID:8770210

  18. Impact of the β-Lactam Resistance Modifier (−)-Epicatechin Gallate on the Non-Random Distribution of Phospholipids across the Cytoplasmic Membrane of Staphylococcus aureus

    PubMed Central

    Rosado, Helena; Turner, Robert D.; Foster, Simon J.; Taylor, Peter W.

    2015-01-01

    The polyphenol (−)-epicatechin gallate (ECg) inserts into the cytoplasmic membrane (CM) of methicillin-resistant Staphylococcus aureus (MRSA) and reversibly abrogates resistance to β-lactam antibiotics. ECg elicits an increase in MRSA cell size and induces thickened cell walls. As ECg partially delocalizes penicillin-binding protein PBP2 from the septal division site, reduces PBP2 and PBP2a complexation and induces CM remodelling, we examined the impact of ECg membrane intercalation on phospholipid distribution across the CM and determined if ECg affects the equatorial, orthogonal mode of division. The major phospholipids of the staphylococcal CM, lysylphosphatidylglycerol (LPG), phosphatidylglycerol (PG), and cardiolipin (CL), were distributed in highly asymmetric fashion; 95%–97% of LPG was associated with the inner leaflet whereas PG (~90%) and CL (~80%) were found predominantly in the outer leaflet. ECg elicited small, significant changes in LPG distribution. Atomic force microscopy established that ECg-exposed cells divided in similar fashion to control bacteria, with a thickened band of encircling peptidoglycan representing the most recent plane of cell division, less distinct ribs indicative of previous sites of orthogonal division and concentric rings and “knobbles” representing stages of peptidoglycan remodelling during the cell cycle. Preservation of staphylococcal membrane lipid asymmetry and mode of division in sequential orthogonal planes appear key features of ECg-induced stress. PMID:26213914

  19. Modeling of closed membrane shapes

    NASA Astrophysics Data System (ADS)

    Penič, S.; Mesarec, L.; Fošnarič, M.; Kralj Iglič, V.; Kralj, S.; Góźdź, W.; Iglič, A.

    2014-12-01

    Closed biological membranes were considered within the spontaneous curvature model. Ground state membrane shapes were compared with Monte Carlo simulations in the thermal equilibrium, where membranes are subject to thermal fluctuations. The results of the two approaches correspond well with each other. The oblate discocyte membrane shapes are obtained in the ground state but can become metastable when thermal fluctuations are taken into account. The nematic ordering in oblate and stomatocyte vesicle membranes was also studied. It was confirmed that the net topological charge on the surfaces with the topology of a sphere was 2. On the oblate vesicle four topological defects, each with charge 1/2, assembled in the region exhibiting the highest Gaussian curvature. On the stomatocyte vesicle with six topological defects, each with charge 1/2, and two topological antidefects, each with charge -1/2, the latter assembled in the region with a negative Gaussian curvature. The position of topological defects is strongly curvature dependent.

  20. Effect of phospholipidic boundary lubrication in rigid and compliant hemiarthroplasty models.

    PubMed

    Foy, J R; Williams, P F; Powell, G L; Ishihara, K; Nakabayashi, N; LaBerge, M

    1999-01-01

    Hemiarthroplasty may benefit from materials which produce lower friction and improved boundary lubrication protection during start-up conditions. The purpose of this study was to evaluate the effect of phospholipidic boundary lubrication in both rigid and compliant hemiarthroplasty. An in vitro model was designed to dissociate the relative contribution of implant material compliance and the presence of phospholipid to the overall friction of a hemiarthroplasty contact using bovine articular cartilage. Normal bovine articular cartilage was articulated against four flat materials using reciprocating motion: (a) borosilicate glass: (b) borosilicate glass coated with dipalmitoylphosphatidylcholine (DPPC); (c) polyurethane (PU) elastomer (Tecoflex SG93A, a medical-grade aliphatic thermoplastic PU, Thermedics Incorporated. Woburn, Massachusetts); and (d) surface-coated PU (Tecoflex SG93A substrate coated with lipid-attracting copolymer poly[methacryloyloxyethyl phosphorylcholine (MPC)-co-butyl methacrylate (BMA)]. Tests were conducted in physiologically simulated tribological conditions for a non-conformal point contact. Friction and lubrication analysis was performed using both static and kinetic coefficients of friction mu measured for each group as a function of time for a sliding distance of up to 60 m. Results showed that the inclusion of supplemental phospholipid, DPPC, on a rigid substrate significantly decreased mu in comparison with the control (cartilage-glass). Additionally, removal of phospholipid components from the articular cartilage surface produced a significantly greater start-up mu in comparison with normal cartilage at the test onset. The use of a material with a lower modulus resulted in lower mu for the entire duration of the test. Polyurethane elastomer coated with the lipid-attracting copolymer, poly(MPC-co-BMA), resulted in the lowest frictional response. As seen in this study, the improvement of low-modulus hemiarthroplasty may involve the

  1. Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

    PubMed Central

    Lehtonen, J. Y.; Kinnunen, P. K.

    1995-01-01

    Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the

  2. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Choubey, Amit

    Biological cell membranes provide mechanical stability to cells and understanding their structure, dynamics and mechanics are important biophysics problems. Experiments coupled with computational methods such as molecular dynamics (MD) have provided insight into the physics of membranes. We use long-time and large-scale MD simulations to study the structure, dynamics and mechanical behavior of membranes. We investigate shock-induced collapse of nanobubbles in water using MD simulations based on a reactive force field. We observe a focused jet at the onset of bubble shrinkage and a secondary shock wave upon bubble collapse. The jet length scales linearly with the nanobubble radius, as observed in experiments on micron-to-millimeter size bubbles. Shock induces dramatic structural changes, including an ice-VII-like structural motif at a particle velocity of 1 km/s. The incipient ice VII formation and the calculated Hugoniot curve are in good agreement with experimental results. We also investigate molecular mechanisms of poration in lipid bilayers due to shock-induced collapse of nanobubbles. Our multimillion-atom MD simulations reveal that the jet impact generates shear flow of water on bilayer leaflets and pressure gradients across them. This transiently enhances the bilayer permeability by creating nanopores through which water molecules translocate rapidly across the bilayer. Effects of nanobubble size and temperature on the porosity of lipid bilayers are examined. The second research project focuses on cholesterol (CHOL) dynamics in phospholipid bilayers. Several experimental and computational studies have been performed on lipid bilayers consisting of dipalmitoylphosphatidylcholine (DPPC) and CHOL molecules. CHOL interleaflet transport (flip-flop) plays an important role in interleaflet coupling and determining CHOL flip-flop rate has been elusive. Various studies report that the rate ranges between milliseconds to seconds. We calculate CHOL flip-flop rates by

  3. Tracking Membrane Protein Association in Model Membranes

    PubMed Central

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S.; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We

  4. The Phospholipid Scramblases 1 and 4 Are Cellular Receptors for the Secretory Leukocyte Protease Inhibitor and Interact with CD4 at the Plasma Membrane

    PubMed Central

    Bouchet, Jérôme; Zarka, Marion; Moura, Ivan C.; Benhamou, Marc; Monteiro, Renato C.; Hocini, Hakim; Madrid, Ricardo; Benichou, Serge

    2009-01-01

    Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention. PMID:19333378

  5. Substitution of ether linkage for ester bond in phospholipids increases permeability of bilayer lipid membrane for SkQ1-type penetrating cations.

    PubMed

    Il'yasova, T M; Rokitskaya, T I; Severina, I I; Antonenko, Y N; Skulachev, V P

    2012-09-01

    Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed. PMID:23157264

  6. Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: roles of membrane proteins, phospholipids and their fatty acid compositions.

    PubMed

    Kraffe, Edouard; Marty, Yanic; Guderley, Helga

    2007-01-01

    Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5 degrees C to 15 degrees C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15 degrees C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5 degrees C to 15 degrees C, the maximal oxidative capacity of mitochondria measured at 15 degrees C increased slightly, but rates at both assay temperatures (5 degrees C and 15 degrees C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15 degrees C were expressed relative to cytochrome a and c(1) levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15 degrees C decreased the ratio of

  7. Identification of a Plasmodium falciparum Phospholipid Transfer Protein*

    PubMed Central

    van Ooij, Christiaan; Withers-Martinez, Chrislaine; Ringel, Alessa; Cockcroft, Shamshad; Haldar, Kasturi; Blackman, Michael J.

    2013-01-01

    Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte. PMID:24043620

  8. Regulation of Membrane Proteins by Dietary Lipids: Effects of Cholesterol and Docosahexaenoic Acid Acyl Chain-Containing Phospholipids on Rhodopsin Stability and Function

    PubMed Central

    Bennett, Michael P.; Mitchell, Drake C.

    2008-01-01

    Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (Ea) was determined from DSC thermograms by four separate methods. Both Tm and Ea varied with bilayer composition. Cholesterol increased the Tm both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered Ea in the absence of DHA, but raised Ea in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The Tm for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (Ea) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability. PMID:18424497

  9. How Do Membranes Respond to Pressure?

    PubMed

    Matsuki, Hitoshi

    2015-01-01

    Bilayers formed by phospholipids are fundamental structures of biological membranes. The mechanical perturbation brought about by pressure significantly affects the membrane states of phospholipid bilayers. In this chapter, we focus our attention on the pressure responsivity for bilayers of some major phospholipids contained in biological membranes. At first, the membrane states and phase transitions of phospholipid bilayers depending on water content, temperature and pressure are explained by using the bilayer phase diagrams of dipalmitoylphosphatidylcholine (DPPC), which is the most familiar phospholipid in model membrane studies. Subsequently, the thermotropic and barotropic bilayer phase behavior of various kinds of phospholipids with different molecular structures is discussed from the comparison of their temperature--pressure phase diagrams to that of the DPPC bilayer. It turns out that a slight change in the molecular structure of the phospholipids produces a significant difference in the bilayer phase behavior. The systematic pressure studies on the phase behavior of the phospholipid bilayers reveal not only the pressure responsivity for the bilayers but also the role and meaning of several important phospholipids existing in real biological membranes. PMID:26174389

  10. A new technique of depositing phospholipid bilayers on quartz surfaces: its use in membrane spin-label studies.

    PubMed

    Kawano, I; Floyd, R A; Sridhar, R

    1981-03-01

    We have developed a new improved technique termed the parallel-beam spattering (PBS) method for depositing phospholipid bilayers on quartz surfaces. This technique involves atomizing the phospholipid mixture with a stream of nitrogen gas and passing this atomized mixture through two orifices separated by a distance to achieve a parallel beam of atomized particles before deposition on the quartz plate. A static electric field can easily be applied to the quartz surface. Also a goniometer of new design has been constructed to allow precise positioning of the deposited phospholipid bilayers with reference to the magnetic field. We have utilized the PBS method to deposit phosphatidylcholine/nitroxyl labeled cholestane mixtures on quartz plates and have found that hydrated bilayers of these mixtures yield ESR spectra with essentially the same characteristics as those obtained using more conventional techniques. The distinct advantage of the new technique for depositing bilayers is that there is no spectral anomaly present which usually is present when the more conventional method of depositing bilayers is used. The spectral anomaly is apparently caused by a portion of the bilayers aligned in directions not directly parallel to the quartz surface. For precision work the spectral anomaly is unacceptable. It is not observed with the new PBS method which has yielded highly reproducible results. PMID:6263962