Showing papers on "Membrane lipids published in 1993"

Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(1-40)] in bilayer membranes

Abstract: We have recently shown that the Alzheimer disease 40-residue amyloid beta-protein [A beta P-(1-40)] can form cation-selective channels when incorporated into planar lipid bilayers by fusion of liposomes containing the peptide. Since A beta P-(1-40) comprises portions of the putative extracellular and membrane-spanning domains of the amyloid precursor protein (APP751), we suggested that the channel-forming property could be the underlying cause of amyloid neurotoxicity. The peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, and we now report that soluble A beta P-(1-40) can also form similar channels in solvent-free lipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome-mediated incorporation, the amyloid channel activity in the patch pipet exhibits multiple conductance levels between 40 and 400 pS, cation selectivity, and sensitivity to tromethamine (Tris). Further studies with A beta P channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective. Amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would be homeostatically dissipative. We therefore interpret these data as providing further support for the concept that cell death in Alzheimer disease may be due to amyloid ion-channel activity.

Cold shock damage is due to lipid phase transitions in cell membranes: A demonstration using sperm as a model

Abstract: When cells are cooled to temperatures above the freezing point of water at rates greater than a few degrees per minute, they sustain irreversible injury. Reduction of this "cold shock" damage could increase the survival of animals and plants at low environmental temperatures and improve the cryopreservation of plant and animal cells. Leakage of solutes across membranes, associated with thermotropic phase transitions in membrane lipids, is thought to be responsible, but this hypothesis has not been tested directly. Using Fourier transform infrared spectroscopy (FTIR), we measured the lipid phase transitions in intact, living sperm, the animal cell in which cold shock has been studied most extensively. A shift in the CH2 absorbance peaks indicates the transition from liquid-crystalline to gel phase. The phase transition in sperm membranes occurred at a lower temperature for a marine shrimp than for the pig. In each case, potassium leakage, which is a hallmark of cold shock damage, increased abruptly near the end of the phase transition. Human sperm are quite resistant to cold shock, and an abrupt lipid phase transition was not detected. This phase behavior is typical of membranes containing a high proportion of cholesterol, and human sperm have an unusually high sterol content. High cholesterol levels are known to stabilize membranes during cooling. Overall, the lipid phase behavior was consistent with the temperature range over which cooling was damaging for pig and shrimp sperm, and the with the extent of damage produced in pig and human sperm. This is the first direct evidence that cold shock results from lipid phase transitions in cell membranes.

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process

Abstract: A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane-associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. However, it is not known whether recycling membrane receptors follow bulk membrane flow or if these proteins are actively sorted from lysosomally directed material because of specific protein sequences and/or structural features. Using quantitative fluorescence microscopy we have determined the endocytic route and kinetics of traffic of the bulk carrier, membrane lipids, to address this issue directly. We show that N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]- sphingosylphosphorylcholine (C6-NBD-SM) in endocytosed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled markers sort from lysosomally destined molecules in peripherally located sorting endosomes, accumulate in a peri-centriolar recycling compartment, and finally exit the cell. Other fluorescently labeled lipids, C6-NBD-phosphatidylcholine and galactosylceramide also traverse the same pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase implies a geometric basis of sorting.

Caveolae and sorting in the trans-Golgi network of epithelial cells.

Abstract: The specific characteristics of cellular membranes are a consequence not only of their protein constituents, but also of their lipid composition. It is obvious that the behavior of a membrane protein is dependent upon its lipid environment. The interplay between proteins and lipids during membrane trafficking is, however, an area of research that has been largely neglected. Interest in the glycolipid-dependent sorting of proteins in membranes is an exception, and has provided a paradigm that demonstrates the importance of these interactions. This phenomenon was first proposed during studies of protein and lipid sorting in simple epithelial cells. The apical and basolateral plasma membrane domains of these cells have not only distinct protein compositions, but the various lipid classes vary in their distribution as well (van Meer and Simons 1988; Simons and van Meer 1988). In particular, the apical plasma membrane is enriched in certain glycolipids. This polarity is in part generated in the trans- Golgi network (TGN), where the proteins and lipids destined for the individual plasma membrane domains are sorted into distinct vesicle carriers (Griffiths and Simons 1986; Wandinger-Ness et al. 1990).

The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803

Abstract: One of the well-characterized phenomena associated with the acclimation of organisms to changes in ambient temperature is the regulation of the molecular motion or "fluidity" of membrane lipids via changes in the extent of unsaturation of the fatty acids of membrane lipids. The enzymes responsible for this process when the temperature is decreased are the desaturases, the activities of which are enhanced at low temperature. To examine whether the change in the fluidity of membrane lipids is the first event that signals a change in temperature, we studied the effect of the Pd-catalyzed hydrogenation of membrane lipids on the expression of the desA gene, which is responsible for the desaturation of fatty acids of membrane lipids in the cyanobacterium Synechocystis PCC6803. The Pd-catalyzed hydrogenation of plasma membrane lipids stimulated the expression of the desA gene. We also found that, for unexplained reasons, the hydrogenation was much more specific to a minor phospholipid, phosphatidylglycerol, than to members of other lipid classes. These results suggest that the organism perceives a decrease in the fluidity of plasma membrane lipids when it is exposed to a decrease in temperature.

Plasma Membrane Lipids Associated with Genetic Variability in Freezing Tolerance and Cold Acclimation of Solanum Species.

Abstract: Simultaneous comparisons were made between a freezing-tolerant, cold-acclimating (CA) wild potato species (Solanum commersonii) and a freezing-sensitive, nonacclimating (NA) cultivated species (Solanum tuberosum). Comparative studies allowed differentiation of plasma membrane lipid changes associated with increased freezing tolerance following CA from lipid changes that can result from metabolic adjustment to reduced temperature during CA. Following CA treatment lipid changes found in both the NA and CA species included a decrease in palmitic acid, an increase in unsaturated to saturated fatty acid ratio, an increase in free sterols, an increase in sitosterol, and a slight decrease in cerebrosides. Lipid changes detected only in the acclimating species included an increase in phosphatidylethanolamine, a decrease in sterol to phospholipid ratio, an increase in linoleic acid, a decrease in linolenic acid, and an increase in acylated steryl glycoside to steryl glycoside ratio. These changes were either absent or opposite in the NA species, suggesting an association of these lipid changes with CA. Furthermore, the lipid changes associated with increased freezing tolerance during CA were distinct from lipid differences between the two species in the NA state.

WIF-B cells: an in vitro model for studies of hepatocyte polarity.

Abstract: We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Lipid headgroup and acyl chain composition modulate the MI-MII equilibrium of rhodopsin in recombinant membranes

Abstract: A current paradigm for visual function centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the trigger for an intracellular enzyme cascade leading to excitation of the retinal rod. We investigated the influences of the membrane lipid composition on this key triggering event in visual signal transduction using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form membrane recombinants in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the lipid headgroups was varied. The ratio of MII/MI produced in these recombinants by an actinic flash at 28 degrees C was studied as a function of pH. The results were compared to the photochemical function observed for rhodopsin in native retinal rod outer segment (ROS) membranes, in total native ROS lipid recombinants, and in dimyristoylphosphatidylcholine (DMPC) recombinants. In membrane recombinants incorporating lipids derived from egg PC, as well as in the total ROS lipids control and the native ROS disk membranes, MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond time scale. The recombinants of rhodopsin with egg PC, either alone or in combination with egg PC-derived phosphatidylethanolamine (PE) or phosphatidylserine (PS), exhibited substantially reduced photochemical activity at pH 7.0. However, all recombinants comprising phospholipids with unsaturated acyl chains were capable of full native-like MII production at pH 5.0, confirming previous results [Gibson, N.J.. & Brown, M.F. (1990) Biochem. Biophys. Res. Commun. 169, 1028-1034]. It follows that energetic constraints on the MI and MII states imposed by egg PC-derived acyl chains can be offset by increased activity of H+ ions. The data reveal that the major effect of the membrane lipid composition is to alter the apparent pK for the MI-MII conformational equilibrium of rhodopsin [Gibson, N.J., & Brown, M.F. (1991) Biochem. Biophys. Res. Commun. 176, 915-921]. Recombinants containing only phosphocholine headgroups exhibited the lowest apparent pK values, whereas the presence of either 50 mol % PE or 15 mol % PS increased the apparent pK. The inability to obtain full native-like function in recombinants having egg PC-derived chains and a native-like headgroup composition indicates a significant role of the polyunsaturated docosahexaenoic acid (DHA) chains (22:6 omega 3) of the native retinal rod membrane lipids. Temperature studies of the MI-MII transition enabled an investigation of lipid influences on the thermodynamic parameters of a membrane protein conformational change linked directly to function.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid absorption: passing through the unstirred layers, brush-border membrane, and beyond.

Abstract: Lipids are components of our diet and luminal secretions, with physicochemical characteristics that determine their digestion and absorption in the gastrointestinal tract Lipids include triglycerides, phospholipids, and cholesterol Dietary lipids contain approximately 97% triglycerides, with small amounts of phospholipids and cholesterol These components are important in cell membrane composition, fluidity, peroxidation, prostaglandin and leukotriene synthesis, and cellular metabolic processes Lipids are implicated in the mechanisms of brain development, inflammatory processes, atherosclerosis, carcinogenesis, aging, and cell renewal Duodenal hydrolysis of dietary lipids and biliary phospholipids and cholesterol is carried out by pancreatic lipase, colipase, phospholipase A2, and cholesterol esterase Bile acid solubilization results in mixed micelles and liposomes, in gel and liquid crystal phases Lipid digestion products pass across the intestinal unstirred water layer For long-chain fatty acids

The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes

Abstract: During invasion of an erythrocyte by a malaria merozoite, an indentation develops in the erythrocyte surface at the point of contact between the two cells. This indentation deepens as invasion progresses, until the merozoite is completely surrounded by a membrane known as the parasitophorous vacuole membrane (PVM). We incorporated fluorescent lipophilic probes and phospholipid analogs into the erythrocyte membrane, and followed the fate of these probes during PVM formation with low-light-level video fluorescence microscopy. The concentration of probe in the forming PVM was indistinguishable from the concentration of probe in the erythrocyte membrane, suggesting that the lipids of the PVM are continuous with and derived from the host cell membrane during invasion. In contrast, fluorescently labeled erythrocyte surface proteins were largely excluded from the forming PVM. These data are consistent with a model for PVM formation in which the merozoite induces a localized invagination in the erythrocyte lipid bilayer, concomitant with a localized restructuring of the host cell cytoskeleton.

Architecture and function

Abstract: Crystallization of Membrane Proteins for X-Ray Analysis. Determination of Cell Surface Polarity by Solid-Phase Lactoperoxidase Iodination. Biochemical Methods to Determine Cell Surface Topography: Part A: Labeling of Oxidized Glycoproteins with 3H-Borohydride Part B: Identification and Separation of Integral Membrane Proteins Using Triton X-114 Part C: Surface-Selective Labeling of Polarized Proteins on Tight Epithelia Using Sulfo-N-Hydroxysuccinimido-Biotin (S-NHS-B). Use of Antipeptide Antibodies for the Isolation and Study of Membrane Proteins: Part A: Preparation of Antibodies Part B: Affinity-Purification of Antipeptide Antibodies Part C: Purification of Membrane Proteins by Immunoaffinity Chromatography Part D: Competitive ELISA for Determining Membrane Protein Topology. The Production of Monoclonal Antibodies to Membrane Proteins: Part A: Production of Hybridoma Cell Lines Part B: Screening for Monoclonal Antibody Production. Purification of a Membrane Protein (Ca2+/Mg2+-ATPase) and Its Reconstitution into Lipid Vesicles. Measurement of Protein-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy. Measurement of Lipid-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy. Determination of the Transverse Topography of Membrane Lipids Using Enzymes and Covalent Labels as Probes. Determination of the Transverse Topography of Membrane Phospholipids Using Phospholipid Transfer Proteins as Tools. Prothrombinase Complex as a Tool to Assess Changes in Membrane Phospholipid Asymmetry. Fluorescent Glycerolipid Probes: Synthesis and Use for Examining Intracellular Lipid Trafficking: Part A: Synthesis of Fluorescent Glycerolipid Probes Part B: Delivery of Fluorescent Glycerophospholipid Probes to Cultured Cells. Synthesis and Use of Spin-Labeled Lipids for Studies of the Transmembrane Movement of Phospholipids. Measurement of Membrane Fluidity and Membrane Fusion with Fluorescent Probes Extraction and Assay of Cyclic Nucleotides. Analysis of G-Proteins Regulating Signal Transduction Pathways: Part A: Identification of G-Proteins Part B: GTPase Studies Part C: Reconstitution of Second Messenger Pathways in Permeabilized Cell Preparations Using GTP Analogs and Receptor Ligands. Assay of Protein Kinases and Protein Phosphorylation: Part A: Protein Kinase-Mediated Phosphorylation Events Part B: Measurement of Protein Kinase Expression Part C: Measurement of Protein Kinase Activity and Translocation. Analysis of Cellular Phosphoinositides and Phosphoinositols: Extraction and Simple Analytical Procedures: Part A: Biosynthesis and Extraction of Phosphoinositides and Phosphoinositols Part B: Separation of Phosphoinositols by Anion-Exchange Chromatography Part C: Separation of Phosphoinositides by Thin-Layer Chromatography Part D: Deacylation of Phospholipids and Separation of Products by Anion-Exchange Chromatography. Analysis of Cellular Phosphoinositides and Phosphoinositols by High Performance Liquid Chromatography: Part A: Sample and Standards Preparation Part B: HPLC Analytical Methods. Cytosolic Free Calcium Measurements in Single Cells Using Calcium-Sensitive Fluorochromes. Membrane Permeabilization with Bacterial Toxins. Measurement of Ion Fluxes and pH Gradients Across Cell Membranes. Ligand Binding and Processing: The Perfused Liver as a Model System. The Binding of Protein-Ligands to Cell-Surface Receptors. Appendix: 1. Density Gradient Media. Appendix: 2. Balanced Salt Solutions. Index.

Biology of halophilic bacteria, Part II. Membrane lipids of extreme halophiles: biosynthesis, function and evolutionary significance.

Abstract: Archaebacteria (archaea) are comprised of three groups of prokaryotes: extreme halophiles, methanogens and thermoacidophiles (extreme thermophiles). Their membrane phospholipids and glycolipids are derived entirely from a saturated, isopranoid glycerol diether, sn-2,3-diphytanylglycerol ('archaeol') and/or its dimer, dibiphytanyldiglyceroltetraether ('caldarchaeol'). In extreme halophiles, the major phospholipid is the archaeol analogue of phosphatidylglycerolmethylphosphate (PGP-Me); the glycolipids are sulfated and/or unsulfated glycosyl archaeols with diverse carbohydrate structure characteristic of taxons on the generic level. Biosynthesis of these archaeol-derived polar lipids occurs in a multienzyme, membrane-bound system that is absolutely dependent on high salt concentration (4 M). The highly complex biosynthetic pathways involve intermediates containing glycerol ether-linked C20-isoprenyl groups which are reduced to phytanyl groups to give the final saturated polar lipids. In methanogens, polar lipids are derived both from archaeol and caldarchaeol, and thermoacidophiles contain essentially only caldarchaeol-derived polar lipids. The function of these membrane polar lipids in maintaining the stability, fluidity and ionic properties of the cell membrane of extreme halophiles, as well as the evolutionary implications of the archaeol and caldarchaeol-derived structures will be discussed.

Protein lateral mobility as a reflection of membrane microstructure

Abstract: The lateral mobility of membrane lipids and proteins is presumed to play an important functional role in biomembranes. Photobleaching studies have shown that many proteins in the plasma membrane have diffusion coefficients at least an order of magnitude lower than those obtained when the same proteins are reconstituted in artificial bilayer membranes. Depending on the protein, it has been shown that either the cytoplasmic domain or the ectodomain is the key determinant of its lateral mobility. Single particle tracking microscopy, which allows the motions of single or small groups of membrane molecules to be followed, promises not only to reveal new features of membrane dynamics, but also to help explain longstanding puzzles presented by the photobleaching studies, particularly the so-called immobile fraction. The combination of the two complementary technologies should measurably enhance our understanding of membrane microstructure.

Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12-lipoxygenases.

Abstract: When arachidonate 12-lipoxygenase purified from porcine leukocytes was incubated aerobically with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, the phospholipid reacted at up to 30% of the rate of a free fatty acid substrate; the esterified arachidonic acid was oxygenated predominantly to the (12S)-12-hydroperoxy product. The porcine leukocyte enzyme was also capable of metabolizing phosphatidylcholine containing esterified (15S)-15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; oxygenation occurred predominantly at the 14R position. Reaction with mitochondrial and endoplasmic membranes of rat liver produced esterified (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid and (13S)-13-hydroperoxy-9,11-octadecadienoic acid as major oxygenation products. Thus, porcine leukocyte 12-lipoxygenase is capable of oxygenating not only free polyenoic fatty acids but also more complex substrates such as phospholipids and biomembranes. In contrast, the human platelet 12-lipoxygenase is almost inactive with these esterified polyenoic fatty acids. In regard to the function of these enzymes, the leukocyte-type of 12-lipoxygenase has similar catalytic activities to the mammalian 15-lipoxygenase and its physiological function may include the structural modification of membrane lipids.

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids.

Abstract: Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.