Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Pressure variation of the lateral diffusion in lipid bilayer membranes

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.     

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.     

An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion channels

The purpose of this study is to develop an apparatus for simultaneous measurement of electrical and spectroscopic parameters of single ion channels. We have combined the single channel recording apparatus with an artificial lipid bilayer and a fluorescence microscope designed to detect single fluorescent molecules. The artificial membranes were formed on an agarose-coated glass and observed with an objective-type total internal reflection fluorescence microscope (TIRFM). The lateral motion of a single lipid molecule (beta-BODIPY 530/550 HPC) was recorded. The lateral diffusion constant of the lipid molecule was calculated from the trajectories of single molecules as D = 8.5 +/- 4.9 x 10(-8) cm(2)/s. Ionic channels were incorporated into the membrane and current fluctuations were recorded at the single-channel level. After incorporation of Cy3-labeled alametithin molecules into the membrane, bright spots were observed moving rather slowly (D = 4.0 +/- 1.6 x 10(-8) cm(2)/s) in the membrane, simultaneously with the alametithin-channel current. These data show the possibility of the present technique for simultaneous measurement of electrical and spectroscopic parameters of single-channel activities.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Photosynthetic energy conversion under extreme conditions--II: the significance of lipids under light limited growth in Antarctic sea ice diatoms

Low photosynthetic active radiation is a strong determinant in the development and growth of sea ice algae. The algae appear to have universal mechanisms to overcome light limitation. One important process, which is induced under light limitation, is the desaturation of chloroplast membrane lipids. In order to discover whether this process is universally valid in sea ice diatoms, we investigated three species coexisting in chemostats illuminated with 15 and 2 micromol photons m(-2) s(-1) at -1 degrees C. Growth under 2 micromol photons m(-2) s(-1) caused a 50% increase in monogalactosyldiacylglycerols (MGDG) thylakoid membrane related 20:5 n-3 fatty acids. This fatty acid supports the fluidity of the thylakoid membrane and therefore the velocity of electron flow, which is indicated by increasing rate constants for the electron transport between Q(A) (first stable electron acceptor) and bound Q(B) (second stable electron acceptor) (11.16 +/- 1.34 to 23.24 +/- 1.35 relative units). Two micromol photons m(-2) s(-1) furthermore resulted in higher amounts of non-lipid bilayer forming MGDG in relation to other bilayer forming lipids, especially digalactosydiacylglycerol (DGDG). The ratio of MGDG:DGDG increased from 3.4 +/- 0.3 to 5.7 +/- 0.3. The existence of bilayer thylakoid membranes with high proportions of non. bilayer forming lipids is only possible when sufficient thylakoid pigment-protein complexes are present. If more thylakoid pigment-protein complexes are present in membranes, as found under extreme light limitation, less bilayer forming lipids such as DGDG are required to stabilize the bilayer structure. Differences in protein contents between both light intensities were not found. Consequently pigment contents which nearly doubled under 2 micromol photons m(-2) s(-1) must be responsible in balancing the potential stability loss resulting from an increase in MGDG:DGDG ratio.     

A semisynthetic 5-n-alkylresorcinol derivative and its effect upon biomembrane properties

MSAR (1-sulfate-3-myristoyl-5-pentadecylbenzene) is a semisynthetic derivative of 5-n-pentadecylresorcinol (C15:0). MSAR exhibits hemolytic activity against sheep erythrocytes with a EH50 value of (35 +/- 1.7) microM. At low concentrations MSAR also exhibits the ability to protect cells against their hypoosmotic lysis. This protective effect is significant as, at 0.1 microM of MSAR, the extent of osmotically induced cell lysis is reduced by approx. 20%. It was demonstrated that the 9-anthroyloxystearic acid signal was most intensively quenched by MSAR molecules, suggesting a relatively deep location of these molecules within the lipid bilayer. MSAR causes an increase of the fluorescence of the membrane potential sensitive probe. This indicates an alteration of the surface charge and a decrease of the local pH value at the membrane surface. At low bilayer content (1-4 mol%) this compound causes a significant increase of the phospholipid bilayer fluidity (both under and above the main phase transition temperature) of dipalmitoylphosphatidylcholine (DPPC) liposomes. At this low content MSAR slightly decreases the main phase transition temperature (T(c)) value. The effects induced in the phospholipid bilayer by higher contents of MSAR molecules (5-10 mol%) make it impossible to determine the T(c) value and to evaluate changes of the membrane fluidity by using pyrene-labeled lipid. MSAR also causes a decrease of the activity of membrane-bound enzymes - red blood cell acetylcholinesterase (AChE) and phospholipase A2 (PLA2). MSAR decreases the AChE activity by 40% at 100 microM. The presence of MSAR in the liposomal membrane induces a complete abolishment of the lag time of the PLA2 activity, indicating that these molecules induce the formation of packing defects in the bilayer which may result from imperfect mixing of phospholipids.     

Insulin-induced changes in mechanical characteristics of lipid bilayers modified by liver plasma membrane fragments

Insulin interaction with BLM with incorporated fragments of rat liver plasma membranes, containing hormone receptors, was studied by determining Young modulus of elasticity of bilayer lipid membranes in direction perpendicular to the surface, E. The presence of membrane proteins in a concentration of 60 micrograms.ml-1 induced a significant decrease in parameter E (to approx. 50%) as compared with values obtained in non-modified membranes during insulin action (concentration interval 10(-11)-10(-9) mol.l-1). The extent of the effect was dependent on the initial phase state of the membrane, on cholesterol content in BLM as well as on membrane proteins concentration in lipid bilayer.     

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.     

Plant sterols: a neutron diffraction study of sitosterol and stigmasterol in soybean phosphatidylcholine membranes

Neutron scattering experiments have been performed on oriented Soybean phosphatidylcholine (SPC) bilayers, containing sitosterol or stigmasterol, two major sterols of plant plasma membranes. Sitosterol and stigmasterol were either protonated or deuterated on position C25 of the lateral chain. Incorporation of sitosterol leads to an increase of the hydrophobic thickness of SPC bilayers of 1.2 and 2 A when present, at 16 and 30 mol%, respectively. On the other hand, no change was observed when stigmasterol is present in the bilayer at its maximal solubility of 16 mol%. These results are in agreement with the fact that sitosterol is more efficient than stigmasterol to order acyl chains of SPC, as already shown with other biophysical techniques. In order to get more insight into the behavior of the lateral chains of the two sterols, the proton-deuterium contrast method was used in order to locate the (2)H25 atoms of the two sterols. For sitosterol, this atom was found close to the center of the bilayer at +/-(1.6+/-0.2 A), with a width, nu=2.5+/-0.5 A. For stigmasterol, the difference profile could be fitted in two different ways: either two possible locations are found at +/-(2.3+/-0.2 A) and +/-(10+/-0.2 A) with the same width, nu=2.5+/-0.5 A or only one broad distribution at +/-(6.1+/-0.3 A), nu=8.5+/-0.7 A. The results are discussed in terms of difference of dynamics for the lateral chain of the two sterols.     

Permeability of bilayer phospholipid membranes to superoxide oxygen radicals

Lecithin monolayer liposomes (1000 A in diameter) loaded with cytochrome c were placed into the external solution, in which O2 superoxide radicals were regenerated by the xanthine-xanthine oxidase system. The penetration of superoxide radicals across the liposomal membranes was followed by cytochrome c reduction in the interval volume of the liposomes. The effects of lipid membrane modifiers and temperature on this process were investigated. The results obtained were used for calculation of the permeability coefficients of bilayer lipid membranes for O(2) (P'O(2) = (7.6 +/- 0.3) . 10(-8) cm . s-1) or HO . 2(P'HO(2) = 4.9 x 10(-4) cm . s-1). The effect of the transmembrane electric potential (concentration gradient of H+, valinomycin) on the permeability of liposomal membranes for the superoxide radical was studied. The superoxide radical was down to penetrate across the bilayer lipid membranes in an unloaded state. Using an intramolecular cholesterol-amphotericin B-complex, the superoxide radicals were shown to penetrate across the bilayer lipid membranes, predominantly via the anionic channels.     

Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide.

The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0, inducing at low pH leakage of contents from vesicles. Leakage from neutral or negatively charged vesicles at pH 5.0 was similar and could be adequately explained by the mathematical model (Parente, R. A., S. Nir, and F. C. Szoka, Jr., 1990. Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry. 29:8720-8728) which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of 10 +/- 2 peptides. Increasing cholesterol content in the membranes resulted in a reduced efficiency of the peptide to induce leakage. Part of the cholesterol effect was due to reduced binding of the peptide to cholesterol-containing membranes. An additional effect of cholesterol was to increase reversibility of surface aggregation of the peptide in the membrane. Results could be explained and predicted with a model that retains the same pore size, i.e., 10 +/- 2 peptides, but includes reversible aggregation of the monomers to form the pore. Resonance energy transfer experiments using fluorescently labeled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol-containing liposomes, thus reducing the efficiency of pore formation.     

Bilayer membrane bending stiffness by tether formation from mixed PC-PS lipid vesicles

Recently, a new approach to measure the bending stiffness (curvature elastic modulus) of lipid bilayer membrane was developed (Biophys. J., Vol. 55; pp. 509-517, 1989). The method involves the formation of cylindrical membrane strands (tethers) from bilayer vesicles. The bending stiffness (B) can be calculated from measurements of the tether radius (Rt) as a function of the axial force (f) on the tether: B = f.Rt/2 pi. In the present report, we apply this method to determine the bending stiffness of bilayer membranes composed of mixtures of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) and POPS (1-palmitoyl-2-oleoyl phosphatidyl serine). Three different mixtures were tested: pure SOPC, SOPC plus 2 percent (mol/mol) POPS, and SOPC plus 16 percent POPS. The bending stiffness determined for these three different lipid mixtures were not significantly different (1.6-1.8 x 10(-12) ergs). Because POPS carries a net negative charge, these results indicate that changes in the density of the membrane surface charge have no effect on the intrinsic rigidity of the membrane. The values we obtain are consistent with published values for the bending stiffness of other membranes determined by different methods. Measurements of the aspiration pressure, tether radius and the tether force were used to verify a theoretical relationship among these quantities at equilibrium. The ratio of the theoretical force to the measured force was 1.12 +/- 0.17.     

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.     

Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.     

Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates

Treatment of synaptic membranes from rat brainstem and spinal cord with the nonionic detergent Triton X-100 at 1-10 microliters/mg protein caused a marked increase in glycine receptor (3H)strychnine binding expressed per mg of residual membrane protein. The effect was maximal (220 +/- 6% of control) at 5 microliters Triton/mg protein, while higher concentrations caused progressive loss of strychnine binding ability of membranes (27 +/- 6% at 25 microliters Triton/mg protein). The increase in strychnine binding caused by low Triton X-100 reflected an increase in membrane Bmax, the kD being unaffected by the treatment. The affinity of glycine analogues for receptor sites was not appreciably affected by the detergent either. The findings suggest an enrichment of the synaptic membrane preparation in glycine receptors, caused by the solubilization by Triton of membrane constituents not related to the receptor sites.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

The effect of nonadec(en)ylresorcinol on the fluidity of liposome and erythrocyte membranes

The effect of alk(en)ylresorcinol homologs (5-(n-nonadecyl)- and 5-(n-nonadecenyl)resorcinol) on the mobility of 5-doxyl- and 12-doxylstearate spin probes incorporated into DMPC, DMPC-cholesterol and erythrocyte membranes was studied. It was found that both homologs affect the properties of hydrophobic environment of the membranes: (1) In DMPC vesicles both homologs induce an increase in the order parameter of 5-doxylstearate at temperatures of Tc and above. (2) At higher concentrations of both homologs a decrease in mobility of the 12-doxylstearate was also observed. (3) In the presence of cholesterol in the liposome membrane the influence of alk(en)ylresorcinols on the mobility of spin probes was much greater, depending on the cholesterol content and the position of the probe in the bilayer. (4) In natural membranes (erythrocyte ghosts) both alkyl- and alkenylresorcinols induced a decrease of mobility in the region of 12-doxylstearate as well as in the region closer to the polar head groups of lipids (5-doxylstearate).     

Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.