Showing papers on "Membrane lipids published in 1979"

Structural order of lipids and proteins in membranes: evaluation of fluorescence anisotropy data

Abstract: The limiting long-time value of fluorescence anisotropy in membranes is correlated with the orientational order parameter, which characterizes the structural anisotropy of membranes. Existing experimental results for diphenylhexatriene in lipid bilayers are evaluated for the order parameter of lipid order. Steady-state measurements of fluorescence anisotropy can provide the order parameter in good approximation. Proteins in a fluid lipid phase increase the lipid order parameter so determined. Upon comparison with the order parameter from deuterium magnetic resonance, it is concluded that proteins increase the order of the surrounding lipids in off-normal directions. Order parameters of protein order obtained from the limiting value of protein fluorescence anisotropy are discussed with respect to the influence of lipid order on protein order.

beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling

Abstract: The beta-adrenergic agonist L-isoproterenol stimulated the enzymic synthesis of phosphatidyl-N-monomethylethanolamine and phosphatidylcholine in rat reticulocyte ghosts containing the methyl donor S-adenosyl-L-methionine. The stimulation was stereospecific, dose-dependent, and inhibited by the beta-adrenergic agonist propranolol. The addition of GTP inside the resealed ghosts shifted the dose-response of phospholipid methylation by L-isoproterenol to the left by 2 orders of magnitude. Direct stimulation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with sodium fluoride or cholera toxin did not increase the methylation of phospholipids. At a concentration of S-adenosyl-L-methionine that stimulates synthesis of phosphatidyl-N-monomethylethanolamine, the activity of isoproterenol-sensitive adenylate cyclase was increased 2-fold without changes in the basal activity of adenylate cyclase and the number of beta-adrenergic receptors. The increase of phospholipid methylation by L-isoproterenol decreased membrane viscosity and increased translocation of methylated lipids. These findings indicate that enhancement of phospholipid methylation by L-isoproterenol decreases membrane microviscosity and thus increases lateral movement of the beta-adrenergic receptors and coupling with adenylate cyclase.

Protection of liposomal lipids against radiation induced oxidative damage.

Abstract: Liposomes were prepared from phospholipids extracted from biological membranes. A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes. The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation. High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation. Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation. The naturally occurring compounds reduced glutathione (GSH) and vitamin E(alpha-T) and the powerful radiation protector cysteamine (MEA). All three molecules could protect the liposomes against peroxidation. The membrane-soluble compound vitamin E was by far the most powerful. About 50 per cent protection was achieved by using 5 X 10(-6) M alpha-T, 10(-4) M GSH and 5 X 10(-4) M MEA. The fatty acid composition of the lipids altered drastically as a result of the irradiation. Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids. Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes.

Erythrocyte membrane lipid reorganization during the sickling process.

Abstract: Summary. In order to study possible alterations in membrane lipids during sickling, we have measured the difference in susceptibility to lipid peroxidation, binding of trinitrobenzenesulfonic acid (TNBS) to aminophospholipids, and fatty acid uptake in cells containing sickle haemoglobin under aerobic and anaerobic conditions. We have also examined TNBS binding in irreversibly sickled cells in an attempt to evaluate the permanent effects of any such alterations. We found that when erythrocytes were sickled by deoxygenation, the susceptibility to lipid peroxidation and binding of TNBS to aminophospholipids was markedly increased, while normal control cells showed no change. These effects appeared to be specific for the sickled state rather than a nonspecific consequence of cell age or the concentration of sickle haemoglobin within the cell. In contrast, fatty acid incorporation into membrane phospholipids, representing potential lipid renewal, was decreased in the sickle state. Cell fractions enriched in irreversibly sickled cells showed increased TNBS labelling in air and only modest rises with anoxia. Taken together, these data imply a rearrangement of membrane lipids during the sickling process and suggest a permanent reorganization of membrane lipids in the irreversibly sickled cell.

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity

Abstract: Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64% The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 04 nmol/mg of protein, or 14% Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4 The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins

Rate limitation of (Na+ + K+)-stimulated adenosinetriphosphatase by membrane acyl chain ordering.

Abstract: A somatic cell mutant (CR1) of the Chinese hamster ovary cell line (CHO-K1) that is defective in the regulation of cholesterol biosynthesis can be grown under conditions in which plasma membranes from these cells display various cholesterol contents and acyl chain order parameters. The (Na+ + K+)-stimulated adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from these cells was shown to vary in activity by a factor of 10 as the order parameter was varied, and the activity exhibited an exponential dependence on this parameter. Under these conditions the number of Na+,K+-ATPase molecules was shown to remain constant by affinity labeling with [gamma-32P]ATP in the absence of Na+. Control experiments showed that alteration in cholesterol content without change in order parameter did not result in altered enzyme activity. It is concluded that, under our conditions, the rate of catalysis by the Na+,K+-ATPase is determined by the order parameter. These studies suggest a physical mechanism by which variation of membrane lipid composition or other factors that determine membrane lipid acyl chain order parameter can result in variation in membrane enzyme activity.

Absence of filipin-sterol complexes from large coated pits on the surface of culture cells.

Abstract: Monolayer cultures of normal or transformed fibroblasts and of liver cells fixed in a glutaraldehyde solution containing 300 microM filipin, a sterol-specific polyene antibiotic, were freeze-fractured to study the distribution of cholesterol within their plasma membranes. Filipin-sterol complexes, recognizable as 25- to 30-nm protuberances scattered in the fracture face of plasma membrane, were absent from invaginations corresponding to large, bristle-coated pits (and possibly also from small, flask-shaped invaginations). These results suggest that invaginating regions on the cell surface are specialized plasma membrane domains with a lower cholesterol content than the surrounding membrane. The localized change in membrane fluidity due to the low cholesterol concentration could play a role in endocytosis.

Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity.

Abstract: In order to manipulate the physical properties of the macrophages membrane, methods were developed which potentiated the incorporation of exogenously supplied fatty acids into membrane lipids. Chromatograms of macrophages which were grown in the presence of a variety of fatty acids demonstrated that exogenously supplied unsaturated fatty acids (palmitoleic, oleic, elaidic, linoleic, linolenic and arachidonic acids) were readily incorporated into the cells and selectively altered the fatty acyl composition of macrophage phospholipids. Up to 38% of the total cellular phospholipids were found to be derived from the exogenously added fatty acid supplements. The incorporation of the different fatty acids into cellular phospholipids had striking effects on cellular phagocytic activity. These effects were found to correlate with the degree of unsaturation, and the cis- or trans-double bond configuration. Thus, macrophage phagocytic ingestion rates of 125I-labelled Shigella flexneri were found to alter by more than 2-fold after the cells were cultivated in the presence of cis unsaturated fatty acids.

Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.

Abstract: The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.

Abstract: The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.

Lipid alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Abstract: The lipid composition of cells of Pseudomonas aeruginosa strains resistant to polymyxin was compared with the lipid composition of cells of polymyxin-sensitive strains as to their content of readily extractable lipids (RELs), acid-extractable lipids, the fatty acid composition of RELs, and the contents of various phospholipids in the REL fraction. The polymyxin-resistant strains had an increased content of RELs, but a decreased phospholipid content. The REL fraction from the polymyxin-resistant strains had an increased content of unsaturated fatty acids accompanied by a decreased content of cyclopropane fatty acids as compared with the fatty acid composition of RELs from polymyxin-sensitive strains. The phosphatidylethanolamine content was greatly reduced in the polymyxin-resistant strains, whereas the content of an unidentified lipid, thought to be a neutral lipid lacking either a phosphate, free amino, or choline moiety, was greatly increased. Cell envelopes of the polymyxin-resistant strains contained reduced concentrations of Mg2+ and Ca2+ as compared with the cell envelopes of polymyxin-sensitive strains. It appears that polymyxin resistance in these strains is associated with a significant alteration in the lipid composition and divalent cation content of the cell envelope.

Effect of a local anaesthetic on hydrocarbon chain order in membranes

Abstract: THE mode of action of anaesthetics has provoked considerable interest and speculation1,2. For tetrodotoxin3, it is clear that direct interaction of the anaesthetic with the sodium channel blocks nerve conduction; in other cases changes in lipid bilayer structure, such as membrane expansion2, fluidus expansion1 or surface-charge modulation4, have been invoked. Recently, a mechanism involving an increase in lipid bilayer thickness has been suggested from results obtained using black film techniques5–7. We have investigated the hydrocarbon chain length of a lecithin bilayer in the presence and absence of the anaesthetic benzyl alcohol8,9 using high-field deuterium nuclear magnetic resonance (NMR) spectroscopy of specifically deuterated lipids10–16, to determine the changes in bilayer structure that occur on addition of the anaesthetic alcohol. We report here that at concentrations used for local anaesthesia there is no change in membrane thickness. By comparison, cholesterol (at 0.3 mol fraction) causes an ∼0.46 nm increase in membrane thickness. We therefore suggest that the amount of solvent (tetradecane) in black lipid membranes, and hence their thickness, may be influenced by the presence of benzyl alcohol and cholesterol5.

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

Abstract: The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.

Abstract: Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3′-dioctadecylindocarbocyanine) had a mean (±SD) diffusion coefficient of (1.7 ± 0.3) × 10-8 cm2/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of ≈3 × 10-10 cm2/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.

A Spin-label Study of the Effect of Gamma Radiation on Erythrocyte Membrane. Influence of Lipid Peroxidation on Membrane Structure

Abstract: Gamma-irradiation of bovine erythrocyte membranes (0.1-4 Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C12 but not at the depth of C5. Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes. On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed. Membrane proteins underwent radiation-induced conformational transitions revealed by maleimide spin label which could be also connected with lipid peroxidation.

Lipid composition of Staphylococcus aureus and its derived L-forms.

Abstract: Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.

The role of lipids in the structure and function of membranes.

Abstract: The increased knowledge of the properties of membrane lipids (Ansell et al, 1973) and of lipid-protein interactions (Singer, 1971; Lenaz, 1973, 1977; Vanderkooi, G, 1974) allows a better understanding of the role of lipids in membrane structure and functions Nevertheless, a unifying picture of such a role is lacking, and it is often tacitly assumed that lipids have different roles; this is indeed the main conclusion emerging from analysis of the literature In fact, lipids in membranes have different functions, affecting enzymic activity positively or negatively, being determinants of permeability properties and transport and being involved in the action of membrane binding sites and receptors Moreover, they are determinants of membrane phenomena involving fusion processes (eg, cell movement, pinocytosis, cell division, cell adhesion, secretion) In such functions, lipids may be specific or not The physical state of a lipid, besides the specific chemical nature of certain groups, appears to be very important in its functions It seems therefore appropriate to assign to lipids many different roles