Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

A specific decrease of the fluorescence depolarization of perylene in muscle membranes from mice with muscular dystrophy

The microviscosity of erythrocyte membranes and muscle microsomes from age matched 6-week old control mice REJ 129 Dy/Dy, and mice with muscular dystrophy REJ 129 dy/dy has been estimated by measuring the fluorescence depolarization of perylene. There was no difference between the erythrocyte membranes. The muscle microsomes from dystrophic animals had about 20% lower values than the controls. The temperature dependence indicated that a transition occurs in both sets of muscle microsomes, but the transition temperature was lower in the dystrophic microsomes. Cholesterol, phospholipid and triglyceride analyses of the membranes showed no difference between the erythrocyte membranes. The largest difference in the muscle microsomes was a two-fold increase in cholesterol level found in the dystrophic microsomes. No simple correlation could be made between the lipid analysis and the microviscosity measurements. Since the change in microviscosity is found in membranes isolated from the tissue primarily affected by the dy gene, we suggest that the change in microviscosity may be important in the development of the disease.     

Anisotropy measurements of intrinsic fluorescence of prenyllipids reveal much higher mobility of plastoquinol than alpha-tocopherol in model membranes

As an alternative to a fluorescent probe approach, the intrinsic fluorescence of reduced forms of prenylquinones has been exploited, which offers a convenient means of determining directly motional properties of these molecules. The steady-state fluorescence anisotropy measurements of plastoquinols (PQH(2)) and alpha-tocopherol (alpha-Toc) incorporated into phospholipid liposomes have been performed. The effect of prenyllipid concentration, PQH(2) side chain length and the composition of the membranes has been studied. For the data interpretation, the fundamental anisotropy of alpha-Toc, PQH(2), ubiquinol-10 and alpha-tocopherolquinol, as well as the angles between the absorption and emission transition moments have been also determined. It was concluded that alpha-Toc shows very low mobility in the lipid bilayer, whereas PQH(2)-9 displays significant motional freedom in dipalmitoylphosphatidylcholine vesicles and even higher in egg yolk lecithin membranes.     

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Phospholipid-bound molecular rotors: synthesis and characterization

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotor's viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.     

Orientation and motion of spin-labels in rabbit small intestinal brush border vesicle membranes

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.     

Interaction of aldolase A with lecithin liposomes

The aldolase A binding to the lecithin liposomes (Kd = 2.4 +/- 0.1 X 10(-3) M) has been shown by the fluorescence and tryptophan phosphorescence at the room temperature. The interaction is accompanied by an increase in the phospholipid bilayer microviscosity, and some conformational changes in the hydrophobic part of the enzyme, pronouncing themselves in Trp-147 environment rigidity, decrease. The observation of membrane viscosity vs. incubation time revealed practically instant enzyme-membrane interaction and no gradual incorporation. The accessibility of the NAD-binding domain of aldolase for NADH in the liposome presence remains unaltered.     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

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.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell.

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Effect of a freeze-thaw cycle on properties of microsomal membranes from wheat

A freeze-thaw cycle to −12°C induced several physical and compositional changes in the microsomal membranes isolated from crown tissue of winter wheat (Triticum aestivum L. cv Frederick). Exposing 7-day-old, nonacclimated seedlings to a single freeze-thaw cycle prevented regrowth of the crown and resulted in increased membrane semipermeability. The phospholipid and protein content of microsomal membranes isolated from the crowns decreased by 70 and 50%, respectively. Microsomal membranes isolated after the lethal freeze-thaw stress, and liposomes prepared from total membrane lipids, exhibited greater microviscosity, measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The number of free thiol groups per milligram membrane protein, measured using the specific fluorescent probe, N-dansylaziridine, decreased after freezing. In contrast, acclimated wheat seedlings which showed increased freezing tolerance, as indicated by survival and ion leakage, suffered almost no effects from the freeze thaw treatment as determined by measurements of membrane microviscosity, phospholipid content, protein content, or danzylaziridine fluorescence. An examination of membranes isolated from frozen tissue showed that most of the changes occurred during the freezing and not during the thawing phase.     

The effect of pressure on the lipid microviscosity and phase transition of lung surfactant

The effect of pressure on the lipid dynamics of the rat lung surfactant was studied in liposomes made of the natural lung surfactant of the rat and of model phospholipid mixture. The determined parameter was the lipid microviscosity, monitored by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Osmotic pressure of up to 47 atm, as well as hydrostatic pressure of up to 1.4 kbar, were applied at a constant temperature. The effect of pressure was monitored by the change in the lipid microviscosity of the system. The maximal change achieved with osmotic pressure at a constant temperature was only 30%. This suggests that the conversion of melted lipid to its solid phase above the lipid critical temperature requires several hundred atmospheres. Similarly, measurements of lipid microviscosity under increased hydrostatic pressure revealed transitions which occurred at above 400 atm. Since such pressures are far beyond the physiological scale, it excludes the possibility that pressure alone can be responsible for a full phase transition of the lung surfactant during respiration. Upon decompression, microviscosity of the examined lipid system was found to return to its original values, confirming the reversibility of the process.     

Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors

The microviscosity of the plasmalemma of protoplasts isolated from rose (Rosa hyb. cv. Golden Wave) petals was measured by fluorescence depolarization. The plasmalemma's microviscosity was found to increase in petals which were allowed to age on cut flowers or after isolation as well as in isolated protoplasts aged in an aqueous medium. Increasing the temperature of the cut flowers or the isolated protoplasts enhanced the increase of the microviscosity of the protoplast plasmalemma. The mole ratio of free sterol to phospholipid was greater in protoplasts isolated from old flowers or in protoplasts aged after isolation than in protoplasts isolated from younger flowers. Microviscosity was greatest when protoplasts were aged at pH 4.4 and in the presence of Ca²⁺. Artificial alterations of the sterol to phospholipid ratio in the protoplasts, induced by treatment with liposomes, caused similar changes in their measured microviscosity.

These findings strongly suggest that the increase in the petal plasmalemma microviscosity with age is associated with an increase in the sterol to phospholipid ratio which results, at least partially, from the activity of endogenous phospholipases.

     

Modulation of membrane receptor endocytosis by chemical effectors of membrane fluidity

Several chemical effectors were used to induce changes in spleen B cell membrane fluidity. Membrane fluidity was monitored by fluorescence polarization analysis of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and cell viability was checked not to be affected by the treatments. Membrane immunoglobulin (Ig) endocytosis by the living B cells with modified or unmodified membranes was quantitatively measured by flow cytometry, using a previously described method (Métézeau et al., 1982, 1984). The kinetics of endocytosis of membrane Ig was not affected by chemical effectors increasing membrane fluidity. On the contrary, increasing membrane microviscosity resulted in the slowing down and eventually the blocking of membrane Ig endocytosis. It is suggested that a step depending on membrane microviscosity is involved in the process of endocytosis; this step may become rate limiting when membranes are artificially rendered or naturally become (i.e. for pathological or particularly differentiated cells) more viscous.     

Permeabilization of phospholipid bilayer membranes induced by gas-liquid flow in an airlift bubble column

The permeability of 5(6)-carboxyfluorescein (CF) through the phospholipid bilayer membranes was measured by using the system in which the CF-containing phospholipid vesicles (liposomes) were suspended in the gas-liquid flow in an external loop airlift bubble column. The airlift was operated at various superficial gas velocities UG up to 2.4 cm/s at 25 and 40 degrees C. The CF-containing liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) had the nominal diameters of 50, 100, and 200 nm. The 50- and 100-nm liposomes were stable at 40 degrees C for 5 h even at a high UG value of 2.4 cm/s based on the observed turbidity of the liposome suspension in the airlift. On the other hand, the 200-nm liposomes were stable at a low UG value of 1.4 cm/s, although a progressive decrease in size of the liposomes was implied at the high UG value of 2.4 cm/s. The permeability coefficient PCF of CF through the lipid membrane of the 100-nm liposomes was significantly increased with increasing UG at a high temperature of 40 degrees C, while at a low temperature of 25 degrees C the PCF value was little dependent on UG. As a typical result on the above liposomes, the PCF value (9.2 x 10(-11) cm/s) at 40 degrees C and UG = 2.4 cm/s in the airlift was more than 15 times larger than that at 25 degrees C in the static liquid corresponding to UG = 0. In addition, the dependence of the PCF value on UG at 40 degrees C became more significant with increasing the size of liposomes suspended. The results obtained revealed that the permeability of the liposome membranes could be regulated by suspending the liposomes in the gas-liquid flow in the airlift without modulating the membrane composition of liposomes.     

Hydrolysable tannins change physicochemical parameters of lipid nano-vesicles and reduce DPPH radical - Experimental studies and quantum chemical analysis

Tannins belong to plant secondary metabolites exhibiting a wide range of biological activity. One of the important aspects of the realization of the biological effects of tannins is the interaction with lipids of cell membranes. In this work we studied the interaction of two hydrolysable tannins: 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) and 1,2-di-O-galloyl-4,6-valoneoyl-β-d-glucose (T1) which had the same number of both aromatic rings (5) and hydroxyl groups (15) but differing in flexibility due to the presence of valoneoyl group in the T1 molecule with DMPC (dimyristoylphosphatidylcholine) lipid nano-vesicles (liposomes). Tannins-liposomes interactions were investigated using fluorescence spectroscopy, differential scanning calorimetry, laser Doppler velocimetry, dynamic light scattering and Fourier Transform Infra-Red spectroscopy. It was shown that more flexible PGG molecules stronger decreased the microviscosity of the liposomal membranes and increased the values of negative zeta potential in comparison with the more rigid T1. Both compounds diminished the phase transition temperature of DMPC membranes, interacted with liposomes via PO groups of head of phospholipids and their hydrophobic regions. These tannins neutralized DPPH free radicals with the stoichiometry of the reaction equal 1:1.The effects of the studied compounds on liposomes were discussed in relation to tannin quantum chemical parameters calculated by molecular modeling.     

Interaction of rabbit muscle aldolase with phospholipid liposomes.

The interaction between rabbit muscle fructose diphosphate aldolase and phospholipid model membranes (liposomes) was studied by measurement of the tryptophan fluorescence of the enzyme. Interaction with liposomes decreases intrinsic fluorescence intensity of the enzyme and shifts the emission wavelength maximum to higher values. The effects appear to be strongly dependent on the nature of the phospholipid polar group and on ionic strength. Also, a reversible modification of specific activity of aldolase upon interaction with liposomes was found. It is postulated that aldolase binds to liposomes mainly by electrostatic interactions and that the binding causes a change in the conformation of the enzyme.