Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18–20 °C (Zimmer, G. and Shirmer, H. (1974) Biochim. Biophys. Acta 345, 314–320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions.In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose.These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90° light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence).In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose.Disc electrophoresis revealed a different binding pattern of the two sugars.It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.     

Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition.

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18-20 degrees C (Zimmer, G. and Schirmer, H. (1974) biochim. Biophys. Acta 345, 314-320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions. In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose. These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90 degrees light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence). In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose. Disc electrophoresis revealed a different binding pattern of the two sugars. It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Thermal transitions of red blood cell deformability. Correlation with membrane rheological properties

Red blood cell deformability has been studied by the initial filtration flow rate as a function of temperature. The well-known transition at 49-50 degrees C (probably due to spectrin denaturation) is shown. Another transition is demonstrated around 18 degrees C (the cell becomes stiffer below this temperature range). The erythrocyte membranes prepared by a mild dialysis technique have the same deformability as intact erythrocytes at room temperature; they also show the same low-temperature transition. No such transition has been found for hemoglobin solutions of viscosity 30 g X dl-1. It is interesting to compare these results with those obtained by other methods which measure the properties of natural or artificial lipid membranes and which also demonstrate a thermal transition at 15-20 degrees C. Therefore, the deformability of intact normal erythrocytes seems to depend mainly on the rheological properties of the membrane.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

Raman spectroscopic study of space structure of membrane proteins and membrane lipids in photodamaged human erythrocyte sensitized by hypocrellin B

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Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Light-induced and osmotically-induced changes in chlorophyll a fluorescence in two Synechocystis sp. PCC 6803 strains that differ in membrane lipid unsaturation

Membranes of wild-type (WT) cells of the cyanobacterium Synechocystis sp. PCC 6803 are abundant in polyunsaturated fatty acids in membrane lipids and thus more fluid than membranes of desA^-/desD^- mutant cells which contain no polyunsaturated fatty acids. Using intact cells we examined the effects of normal and chilling temperatures on membrane fluidity-dependent properties. We probed the thylakoid membranes by inducing light/dark acclimative changes in chlorophyll a (Chl a) fluorescence; and we probed the plasma membranes either by suppressing the Chl a fluorescence of light-acclimated cells under hyper-osmotic conditions, or by measuring the electric conductivity of cell suspensions. Thylakoid membranes of mutant cells undergo reversible thermotropic transition between 19 °C and 22 °C (midpoint at 20.5 °C). No analogous transition was detected in the thylakoid membranes of WT cells in the temperature range from 2 to 34 °C. Plasma me mbranes of both WT and mutant cells did not experience thermotropic transition in the temperature range from 2 °C to 34 °C as detected either fluorimetrically or by means of electric conductivity. Hyper-osmotic conditions caused fast transient fluorescence quenching in WT cells at 34 °C, but not at 14 °C, and not in mutant cells at either 34 °C or 14 °C. This transient quenching sensed probably the higher fluidity of the plasma membranes of WT cells. Hyper-osmotic media and dark acclimation had similar effects on the 77 K fluorescence of Synechocystis cells: they suppressed the ratio of photosystem II fluorescence to photosystem I fluorescence.     

Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15°C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16°C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15°C for cucumber (Cucumus sativus L.) fruit, at 14°C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15°C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.     

Irradiation increases proteolysis in erythrocyte ghosts: A spin label study

Summary X- and gamma-irradiation of human erythrocyte membranes (250–1000 Gy) was found to decrease the ratio of weakly to strongly immobilized signal height of membrane-bound maleimide spin label (Mal-6). Subsequent incubation of spin-labeled membranes at ambient temperature (21 °C) induced a progressive increase in this ratio, faster for membranes irradiated with low doses which was hampered by protease inhibitors. These results demonstrate that ionizing radiation stimulates proteolysis of erythrocyte membrane proteins by membrane-associated proteases.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Fluidity of human erythrocyte membrane and effect of chlorpromazine on fluidity and phase separation of membrane

The fluidity of human erythrocyte membrane, and the effect of chlorpromazine at prelytic and lytic concentrations on the fluidity have been studied by using three kinds of fatty acid spin labels and measuring the temperature dependence of Mg2+-ATPase activity. The Arrhenius plot of the apparent rotational correlation time, tau c, for probes I(12,3) and I(5,10) showed an abrupt discontinuity at about 30 degrees C, and the plot for I(1,14) at 25 degrees C, indicating that a large difference in the fluidity exists between the interior and the outer surface of the lipid bilayer. The portions of the fatty acid chain near the ten carbon bond lengths removed from the bilayer surface became more fluid by chlorpromazine treatment; there was a decrease in the break point to around 26 degrees C following treatment with 0.6 or 1 mM of the drug. Two breaks at 21 and 30 degrees C in the Arrhenius plot of the Mg2+-ATPase activity were observed in normal erythrocyte membrane. The activation energy of the Mg2+-ATPase reaction has the values of 3.0 and 22.1 kcal/mol above the upper break and below the lower break, respectively. The drug exposure induced only a slight shift in the break temperatures, while the treatment significantly enhanced the associated activation energies of the reaction. These results suggest that the boundary phospholipids of the Mg2+-ATPase in the membrane are probably more rigid than the bulk lipids.     

C-NMR detection of lipid polymorphism in model and biological membranes

1. 1. The application of the ¹³C-NMR technique to the study of lipid polymorphism is described for various model and biological membranes.

2. 2. The ¹³C-NMR line-width of various resonances of the lipid molecule are sensitive to the bilayer hexagonal and the bilayer ‘isotropic’ phase transition. The latter transition in some cases is accompanied by the occurrence of lipidic particles as detected by freeze-fracturing. Thus, specific ¹³C-labeling experiments allow the study of the individual phase behaviour of lipids in mixed lipid systems.

3. 3. In diet experiments using rats, the choline group of phosphatidylcholine present in erythrocyte, endoplasmic and sarcoplasmic reticulum membranes could be specifically ¹³C-labeled. The ¹³C line-widths of the resonance from the erythrocyte are typical for a lamellar arrangement of the membrane lipids. In strong contrast, the line-width observed at 37°C for the endoplasmic and sarcoplasmic reticulum membranes is much smaller, typical of the isotropic phases observed in model membranes. In isolated rat liver microsomes and liver slices, the ¹³C line-width is strongly temperature dependent. At lower temperatures the line-widths strongly increase towards values typical of lipids in a bilayer structure.

Wheat mitochondria: oxidative activity and membrane lipid structure as a function of temperature

Mitochondrial oxidative activity and membrane lipid structure of two wheat (Triticum aestivum L.) cultivars were measured as a function of temperature. The Arrhenius activation energy for the oxidation of both succinate and α-ketoglutarate was constant over the temperature range of 3 to 27 C. The activation energy for succinate-cytochrome c oxidoreductase activity was also constant over the same temperature range. The concentration of mitochondria in the reaction, the degree of initial inhibition of state 3 respiration, and the time after isolation of mitochondria were each shown to be capable of causing a disproportionate decrease in the rate of oxidation at low temperatures which resulted in an apparent increase in the activation energy of oxidative activity. Using three spin-labeling techniques, wheat membrane lipids were shown to undergo phase changes at about 0 C and 30 C. It is concluded that the membrane lipids of wheat, a chillingresistant plant, undergo a phase transition similar to the transition observed in the membrane lipids of chilling-sensitive plants. For wheat, however, the transition is initiated at a lower temperature and extends over a wider temperature range.