Showing papers on "Membrane lipids published in 1985"

The Enzymes of Biological Membranes

Abstract: of Volume 1.- 1. Electron Microscopy of Biological Membranes.- I. Introduction.- II. Methods Used for Studying Biological Membranes in the Electron Microscope.- A. Sectioning.- B. Negative Staining.- C. Freeze-Etching and Freeze Fracturing.- D. Split Membrane Technique.- E. Immunoelectron Microscopy.- F. Colloidal Gold Marker.- G. Cryoelectron Microscopy.- H. Image Processing and Three-Dimensional Structure Determination.- References.- 2. Associations of Cytoskeletal Proteins with Plasma Membranes.- I. Introduction.- II. The Components of the Cytoskeleton.- A. Actin.- B. Microtubules.- C. Intermediate Filaments.- D. The Role of ?-Actinin.- III. Cytoskeletal Functions.- IV. The Erythrocyte Membrane Skeleton: A Completely Membrane Associated Cytoskeleton.- A. Composition of the Erythrocyte Membrane Skeleton.- B. Spectrin.- C. Actin.- D. Polypeptides 4.1 and 4.9.- E. Ankyrin.- F. Associations between Spectrin, Actin and Band 4.1.- G. Ultrastructure of the Membrane Skeleton.- V. Cytoskeletal Involvement in Cell-Substratum Associations.- A. Focal Adhesions.- B. The Role of ?-Actinin and Vinculin in Focal Adhesions.- C. Association of Vinculin with F-Actin.- D. Effects of Cell Transformation on Focal Adhesions.- E. The Relationship of Focal Adhesions with the Extracellular Matrix.- F. The Formation of Focal Adhesions.- G. The Function of Focal Adhesions.- VI. Cytoskeletal-Membrane Interactions in Microvilli of the Intestinal Brush Border.- VII. Membrane-Associated Cytoskeletal Elements and the Control of Cell Surface Receptor Dynamics.- A. Capping.- B. Endocytosis.- C. Cell Surface Receptor Topography and Mobility.- VIII. Summary.- References.- 3. Cell Coupling.- I. Introduction.- II. Which Molecules Diffuse from Cell to Cell.- A. Electrical Coupling.- B. Cell-to-Cell Diffusion of Ions.- C. Molecular Probes of Cell-to-Cell Coupling.- D. Metabolic Coupling.- E. Variability in Channel Permselectivity.- F. Asymmetry of Channel Permeability.- III. How Molecules Diffuse for Cell-to-Cell.- A. Gap Junction Architecture.- B. Structure of Cell-to-Cell Channels.- C. What Keeps Gap Junction Particles Aggregated.- D. Gap Junction Composition.- IV. How Cell-to-Cell Diffusion of Molecules is Regulated.- A. Uncoupling Agents.- B. Is Uncoupling a Graded Phenomenon?.- C. How to Enhance Coupling or Inhibit Uncoupling.- D. Change in Junction Structure with Uncoupling.- E. Hypotheses on Channel Closing Mechanisms.- F. Is Calmodulin Involved in the Regulation of Cell-to-Cell Coupling?.- References.- 4. Lipid Polymorphism and Membrane function.- I. Introduction.- II. Membrane Lipid Polymorphism: Technical Aspects.- III. Phase Preferences of Membrane Lipids.- IV. The Hexagonal HII Phase.- V. Modulation of Membrane Lipid Polymorphism.- A. One Lipid Systems.- B. Mixed Lipid Systems.- C. Lipid-Protein and Lipid-Peptide Interactions.- VI. "Isotropic" Lipid Structures and Lipid Particles.- VII. The Shape Concept, a Rationale for Lipid Polymorphism.- VIII. Functional Aspects of Lipid Polymorphism.- A. Fusion.- B. Transport.- C. Protein Insertion and Transport.- IX. Lipid Structure in Biological Membranes.- A. Erythrocyte Membrane.- B. Endoplasmic Reticulum (Microsomes).- C. The Inner Mitochondrial Membrane.- D. Bacterial Membranes.- E. Rod Outer Segment (ROS).- F. Chloroplast and Prolamellar Body.- G. Tight Junction.- X. Concluding Remarks.- References.- 5. Intrinsic Protein-Lipid Interactions in Biomembranes.- I. Introduction.- II. Properties of Biomembrane Components.- A. Lipids.- B. Proteins.- III. Lipid Composition and Enzyme Activity.- IV. Specificity of Protein-Lipid Interactions.- V. Distribution of Proteins in Membranes.- VI. Perturbation of Lipid Dynamics by Intrinisic Proteins.- A. NMR and EPR Spectroscopy.- B. Fluorescence Depolarization.- C. Range of the Perturbation.- VII. The Effect of Protein on Lipid Conformation.- A. Acyl Chain Region.- B. Glycerol Backbone Region.- C. Polar Region.- VIII. The Influence of Lipids on Protein Conformation.- IX. Diffusion of Membrane Components.- A. Lateral Diffusion.- B. Rotational Diffusion of Proteins.- X. Summary.- References.- 6. On the Molecular Structure of the Gramicidin Transmembrane Channel.- I. Introduction.- A. Primary Structure.- II. Planar Lipid Bilayer Transport Studies.- A. Phenomenology of Channel Transport.- B. Structural Implications of the Multiplicity of Single-Channel Conductances.- C. Structural Implications of Current/Voltage Curves.- D. Structural Deductions from Derivatives and Analogs.- III. Spectroscopic Characterization of the Lipid Incorporated Channel State.- A. Criteria for the Channel State in Lysolecithin Structures.- B. Relationship between the Lysolecithin-Gramicidin Heat Incorporated Channel State and the State of Gramicidin in Lipid Vesicles.- C. Orientation of Gramicidin Chains in the Lipid Bilayer.- D. Determining the Channel Conformation from Ion-Induced Carbonyl Carbon Chemical Shifts.- References.- 7. Conventional ESR Spectroscopy of Membrane Proteins: Recent Applications.- I. Introduction.- II. The Time Scale of Phospholipid Exchange at the Boundary of Non Aggregated Intrinsic Proteins.- III. Lipids Trapped between Protein Aggregates or Protein Oligomers.- IV. Specificity of Lipid-Protein Interactions as Investigated with Spin Labels.- A. 1st Approach: Estimation of the Relative Percentage of Immobilized Component.- B. 2nd Approach: Spin-Spin Interaction between Nitroxide Radicals.- V. Interactions between Extrinsic Proteins and Lipids.- A. Protein Penetration.- B. Protein Induction of Lateral Phospholipid Separation.- C. Protein Induction of Transverse Phospholipid Separation.- VI. Other Applications of Conventional ESR Spectroscopy to the Investigation of Membrane-Bound Enzymes.- A. Measurement of Surface Potentials and Intermembrane Potentials.- B. Conformation of Membrane-Bound Enzymes.- References.- 8. Saturation Transfer EPR Studies of Microsecond Rotational Motions in Biological Membranes.- I. Introduction.- II. ST-EPR Methodology.- A. General Principles of ST-EPR.- B. Methodology Used in Most Published Applications.- C. Recent Developments in ST-EPR Methodology.- III. Membrane-Bound Enzymes.- A. Sarcoplasmic Reticulum Calcium Transport ATPase.- B. Mitochondrial Electron Transport Chain.- C. Cytochrome P-450.- D. Glyceraldehyde-3-Phosphate Dehydrogenase.- IV. Other Membrane Proteins.- A. Rhodopsin.- B. Acetylcholine Receptor.- C. Red Blood Cell Membranes.- V. Lipid Probes.- VI. Summary.- References.- 9. Dye Probes of Cell, Organelle, and Vesicle Membrane Potentials.- I. Introduction.- II. Types of Potential Sensitive Dyes.- III. Slow Dyes.- A. Mechanism of Slow Dyes.- B. Examples of the Use of Slow Dyes.- IV. Fast Dyes.- A. General Properties.- B. Examples of the Uses of Fast Dyes.- References.- 10. Selective Covalent Modification of Membrane Components.- I. Introduction.- A. Aim and Purpose of Selective Covalent Modification.- B. Biological Membranes as Reactants in Chemical Reactions.- C. Selectivity-Promoting Factors in Membrane Labeling Studies.- II. Covalent Modification of Lipid Components.- A. Lipid Polar Head Group Modification.- B. Lipid Labeling Within the Apolar Membrane Phase.- III. Selective Covalent Modification of Protein Components.- A. Protein Modification Attained by Polar Reagent-Membrane Interaction.- B. Hydrophobic Labeling of Membrane Protein Components.- IV. Information Acquired through Selective Modification.- A. Membrane Structure: Sidedness, Asymmetry and Protein Topography.- B. Membrane Protein Function and Mechanism.- References.- 11. Calcium Ions, Enzymes, and Cell Fusion.- I. Introduction.- II. The Fusion of Myoblasts.- A. Dependence on Ca2+.- B. Some Recent Developments.- III. General Hypotheses: Ca2+, Phospholipids and Membrane Fusion.- A. Ca2+ and ATPase Activity.- B. Phase Separations of Membrane Lipids.- C. Nonbilayer Structures.- IV. Cell Fusion and Vesicle Fusion without Ca2+.- A. Cell Fusion.- B. Vesicle Fusion.- V. Concluding Comments.- References.- 12. Role of Membrane Fluidity in the Expression of Biological Functions.- I. Introduction.- II. Meaning and Measurement of Membrane Fluidity.- A. Definition of Fluidity and Viscosity of Ordinary Liquids.- B. Measurement of Membrane Fluidity through Fluorescence Anisotropy.- C. Measurement of the Conformation (Order) and Dynamics (Fluidity) of Membranes by NMR and ESR.- D. Limitations of the DPH Technique for Measuring Fluidity.- III. Factors that Influence Membrane Fluidity.- A. Lipid Composition.- B. Protein ard Boundary Lipid.- C. pH.- D. Calcium.- E. Salt Concentration.- IV. Mechanisms by which Membrane Fluidity Influences Membrane Functions.- V. Role of Membrane Fluidity in Some Membrane Functions.- A. Effects of Cholesterol.- B. Anesthetics.- C. Aging.- D. Cell Growth and Differentiation.- References.- 13. Rotational Diffusion of Membrane Proteins: Optical Methods.- I. Historical Background.- II. Physical Model for Rotational Diffusion of a Membrane Protein.- III. Physical Principles of Photoselection.- IV. Intrinsic and Extrinsic Probes.- V. Time-Resolved and Steady-State Methods.- VI. Linear Dichroism.- VII. Delayed Fluorescence.- VIII. Phosphorescence.- IX. Fluorescence Depletion.- X. Applications.- XI. Prospects.- References.

Intrinsic curvature hypothesis for biomembrane lipid composition: a role for nonbilayer lipids

Abstract: A rationale is presented for the mix of "bilayer" and "nonbilayer" lipids, which occurs in biomembranes. A theory for the L alpha-HII phase transition and experimental tests of the theory are reviewed. It is suggested that the phase behavior is largely the result of a competition between the tendency for certain lipid monolayers to curl and the hydrocarbon packing strains that result. The tendency to curl is quantitatively given by the intrinsic radius of curvature, Ro, which minimizes the bending energy of a lipid monolayer. When bilayer (large Ro) and nonbilayer (small Ro) lipids are properly mixed, the resulting layer has a value of Ro that is at the critical edge of bilayer stability. In this case, bilayers may be destabilized by the protein-mediated introduction of hydrophobic molecules, such as dolichol. An x-ray diffraction investigation of the effect of dolichol on such a lipid mixture is described. This leads to the hypothesis that biomembranes homeostatically adjust their intrinsic curvatures to fall into an optimum range. Experimental strategies for testing the hypothesis are outlined.

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

Abstract: The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A2, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A2 activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.

Adaptation of the membrane lipids of a deep-sea bacterium to changes in hydrostatic pressure

Abstract: The fatty acid composition of the cell membrane of the barophilic marine bacterium CNPT3 was found to vary as a function of pressure. Greater amounts of unsaturated fatty acids were present in bacteria growing at higher pressures. The results suggest adaptations in the membrane lipids to environmentally relevant pressures. This response to pressure appears to be analogous to temperature-induced membrane adaptations observed in other organisms.

Ethanol and membrane lipids.

Abstract: Although ethanol is known to exert its primary mode of action on the central nervous system, the exact molecular interaction underlying the behavioral and physiological manifestations of alcohol intoxication has not been elucidated. Chronic ethanol administration results in changes in organ functions. These changes are reflective of the adaptive mechanisms in response to the acute effects of ethanol. Biophysical studies have shown that ethanol in vitro disorders the membrane and perturbs the fine structural arrangement of the membrane lipids. In the chronic state, these membranes develop resistance to the disordering effects. Tolerance development is also accompanied by biochemical changes. Although ethanol-induced changes in membrane lipids have been implicated in both biophysical and biochemical studies, measurements of membrane lipids, such as cholesterol content, fatty acid unsaturation, phospholipid distribution, and ganglioside profiles, have not produced conclusive evidence that any of these parameters are directly involved in the action of ethanol. On the other hand, there is increasing evidence indicating that although ethanol in vitro produces a membrane-fluidizing effect, the chronic response to this effect is not to change the membrane bulk lipid composition. Instead, changes in membrane lipids may pertain to small metabolically active pools located in certain subcellular fractions. Most likely, these lipids are involved in important membrane functions. For example, the increase in PS in brain plasma membranes may provide an explanation for the adaptive increase in synaptic membrane ion transport activity, especially (Na,K)-ATPase. There is also evidence that the lipid pool involved in the deacylation-reacylation mechanism (i.e., PI and PC with 20:4 groups) is altered after ethanol administration. An increase in metabolic turnover of these phospholipid pools may have important implications for the membrane functional changes. Obviously, there are other lipid-metabolizing enzyme systems that may exert similar effects but have not yet been investigated in detail. From the results of these studies, it is concluded that the multiple actions of ethanol are associated with changes in enzymic systems important in the functional expression of the membranes.

Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells.

Abstract: We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells. The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of 14C-labelled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cells, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling. The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.

In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides

Abstract: Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

Release of arachidonate from membrane phospholipids in cultured neonatal rat myocardial cells during adenosine triphosphate depletion. Correlation with the progression of cell injury.

Abstract: The present study utilized a cultured myocardial cell model to evaluate the relationship between the release of arachidonate from membrane phospholipids, and the progression of cell injury during ATP depletion. High-energy phosphate depletion was induced by incubating cultured neonatal rat myocardial cells with various combinations of metabolic inhibitors (deoxyglucose, oligomycin, cyanide, and iodoacetate). Phospholipid degradation was assessed by the release of radiolabeled arachidonate from membrane phospholipids. In this model, the current study demonstrates that (a) cultured myocardial cells display a time-dependent progression of cell injury during ATP depletion; (b) the morphologic patterns of mild and severe cell injury in the cultured cells are similar to those found in intact ischemic canine myocardial models; (c) cultured myocardial cells release arachidonate from membrane phospholipids during ATP depletion; and (d) using two separate combinations of metabolic inhibitors, there is a correlation between the release of arachidonate, the development of severe cellular and sarcolemmal damage, the release of creatine kinase into the extracellular medium, and the loss of the ability of the myocardial cells to regenerate ATP when the metabolic inhibitors are removed. Thus, the present results suggest that during ATP depletion, in cultured neonatal rat myocardial cells, the release of arachidonate from myocardial membrane phospholipids is linked to the development of membrane defects and the associated loss of cell viability.

Role of Membrane Lipids and Membrane Fluidity in Thermosensitivity and Thermotolerance of Mammalian Cells

Abstract: The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.

Membrane disordering by anesthetic drugs: relationship to synaptosomal sodium and calcium fluxes.

Abstract: The effects of membrane perturbants (ethanol, pentobarbital, chloroform, diethylether, phenytoin, cis-vaccenic acid methylester, and cis-vaccenoyl alcohol) on the lipid order of mouse brain synaptic plasma membranes (SPM) were tested by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. The compounds decreased the fluorescence polarization of both probes, indicating that they disordered the membrane lipids. The decrease in polarization was, however, greater for DPH than for TMA-DPH, suggesting a greater effect on the membrane core than on the membrane surface. The voltage-dependent uptake of 24Na and 45Ca was studied in isolated mouse brain synaptosomes as a measure of membrane function. All of the compounds inhibited sodium influx, and their potencies for decreasing sodium uptake and fluorescence polarization of DPH were linearly correlated (r = 0.91). The relationship between changes in sodium influx and TMA-DPH polarization was less consistent (r = 0.66). Synaptosomal calcium uptake was inhibited by most, but not all, of the perturbants, but this inhibition was poorly correlated with changes in fluorescence polarization of DPH (r = 0.36) or TMA-DPH (r = 0.26). These results indicate that the function of synaptic sodium channels is correlated with lipid order in the hydrophobic core of the membrane and that the inhibitory effects of intoxicant-anesthetic drugs on neuronal sodium fluxes may be the result of their capacity to disorder these lipids. In contrast, the effects of drugs on voltage-dependent calcium channels were not clearly related to the capacity of these agents to disorder membrane lipids.

Carotenoid-containing outer membrane of Synechocystis sp. strain PCC6714.

Abstract: Outer membranes, free of cytoplasmic or thylakoid membranes and peptidoglycan components, were obtained from Synechocystis sp. strain PCC6714. Electron microscope studies revealed double-track outer membrane vesicles with a smooth-appearing exoplasmic surface, an exoplasmic fracture face covered by closely packed particles and a corresponding plasmic fracture face with regularly distributed holes. Lipopolysaccharide, proteins, lipids, and carotenoids were the constituents of the outer membrane of Synechocystis sp. PCC6714. Twelve polypeptides were found in outer membrane fractions, among them two dominant outer membrane proteins (Mrs, 67,000 and 61,000). Lipopolysaccharide-specific components were GlcN and an unidentified heptose. Outer membrane lipid extracts contained phosphatidylglycerol, sulfolipid, phosphatidylcholine, and unknown lipids. The carotenoids, myxoxanthophyll, related carotenoid-glycosides, zeaxanthin, echinenone, and beta-carotene were found to be true constituents of the outer membrane of Synechocystis sp. PCC6714.

Chronic ethanol increases liver plasma membrane fluidity

Abstract: Purified plasma membrane fractions of cultured well-differentiated Reuber H35 hepatoma cells were studied after growth in the presence or absence of ethanol. Growth of cells in the presence of ethanol significantly increased plasma membrane 5'-nucleotidase activity but did not influence sodium-potassium adenosinetriphosphatase activity. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from cells grown in 80 mM ethanol for 3 weeks, compared to controls. Decreased polarization of DPH in plasma membranes was observed after 3-weeks growth of cells in as little as 1 mM ethanol. A 1-h exposure to 80 mM ethanol had no effect. Altered DPH polarization was due to a decrease in the order parameter of the probe. The rotational correlation time of the probe was virtually unchanged. Chronic ethanol treatment of cells did not alter the polarization of the membrane surface probe trimethylammoniodiphenylhexatriene. Plasma membranes from cells grown in 80 mM ethanol had decreased contents of both phospholipid and unesterified cholesterol, but the cholesterol to phospholipid ratio was unchanged. The percentages of sphingomyelin and phosphatidylserine in plasma membrane phospholipids were significantly decreased after ethanol treatment, while the phosphatidylcholine/sphingomyelin ratio was increased by 42%. Vesicles prepared from total plasma membrane lipids of ethanol-treated cells, as well as vesicles prepared from polar lipids alone, showed the same alterations in DPH polarization as did plasma membranes. The importance of ethanol metabolism in the observed plasma membrane changes was demonstrated in two ways.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin-label studies of lipid-protein interactions in (Na+,K+)-ATPase membranes from rectal glands of Squalus acanthias.

Abstract: Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from the rectal gland of Squalus acanthias have been studied by using spin-labeled lipids in conjunction with electron spin resonance (ESR) spectroscopy. Lipid-protein associations are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component which corresponds very closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled lipids whose motion is very significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A stoichiometry of approximately 66 lipids per 265 000-dalton protein is found for the motionally restricted component of those spin-labeled lipids (e.g., phosphatidylcholine) which show least specificity for the protein. This corresponds approximately to the number of lipids which may be accommodated within the first shell around the alpha 2 beta 2 protein dimer. A selectivity of the various spin-labeled lipids for the motionally restricted component associated with the protein is found in the following order: cardiolipin greater than phosphatidylserine approximately stearic acid greater than or equal to phosphatidic acid greater than phosphatidylglycerol approximately phosphatidylcholine approximately phosphatidylethanolamine approximately androstanol.

Interaction of lipid peroxidation and calcium in the pathogenesis of neuronal injury.

Abstract: The interactions between lipid peroxidation and calcium in mediating damage to central nervous system membranes have been examined in several in vitro systems. Using isolated rat brain synaptosomes, brain mitochondria, or cultured fetal mouse spinal cord neurons, Ca2+ was found to markedly enhance lipid peroxidation-induced disruption of membrane function. Gamma-aminobutyric acid (GABA) uptake by synaptosomes was inhibited 25% by either lipid peroxidation (induced with xanthine and xanthine oxidase) or Ca2+ alone, whereas inhibition was 46% with their combination. Ca2+ enhancement of lipid peroxidation-induced damage to synaptosomes was intensified by the Ca2+ ionophore, A23187, and was partially blocked by the Ca2+ channel blocker, verapamil. Similarly, inhibition of state 3 respiration in isolated rat brain mitochondria was observed with Ca2+ and a free radical generating system (xanthine and xanthine oxidase) under conditions where either insult alone failed to cause detectable damage. Na+,K+-ATPase activity of cultured fetal mouse spinal cord neurons was inhibited 32% when cells were incubated for 30 minutes in the presence of both A23187 and a free radical generating system. However, Na+,K+-ATPase was not affected during a 30 minute incubation with either A23187 or radical generating system alone. In further studies, peroxidation of rat brain synaptosomes by ferrous iron (Fe2+) and H2O2 was coupled with a rapid and large (2-7-fold) uptake of Ca2+ by synaptosomes. Fe2+ also enhanced Ca2+ uptake by spinal cord neurons in culture, an effect that was coincident with peroxidation of neuronal membranes and the release of arachidonic acid from cells. Iron-induced Ca2+ uptake was blocked by high concentrations of either desferrioxamine or methylprednisolone, whereas Ca2+ channel blockers did not affect Ca2+ uptake induced by Fe2+. Finally, peroxidation of membrane lipids by Fe2+ was stimulated by Ca2+. Concentrations of Ca2+ as low as 10(-9) M increased peroxidation reactions within brain synaptosomal membranes. The results of these studies indicate that lipid peroxidation and Ca2+ can synergistically act to damage biologic membranes. The findings suggest that Ca2+ and lipid peroxidation cannot be considered as separate entities in the pathophysiology of CNS trauma. A hypothesis proposing an inseparable interplay between lipid peroxidation and Ca2+ in the pathogenesis of traumatic and ischemic cell injury is presented.

7 Lipid Analysis and the Relationship to Chemotaxonomy

Abstract: Publisher Summary This chapter discusses lipid analysis and the relationship to chemotaxonomy. All cells have a cytoplasmic membrane that serves to form a boundary between itself and the exterior world. The composition of membranes is roughly 50% lipids and 50% non-lipids. The lipid portion contains a half-dozen or more different classes of lipids with each consisting of individual molecular species that have a half-dozen or more structural variations. The relative percentage composition of individual lipids and the lipid structuralization are generally influenced by age of the cells and other environmental parameters. The basic identities of biomembrane lipids have been organized and a seemingly coherent picture has emerged. The majorities of all lipids are membrane associated and present as a complexed integration of both polar and non-polar lipids. To disrupt the hydrogen bonding, electrostatic forces, and interfacial tensions so as to free the lipids, a mixture of solvents with comparable polarity ranges are required.