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.     

Imaging human epithelial properties with polarized light-scattering spectroscopy.

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering.

A laser light source and a digital autocorrelator were employed in the study of the molecular dyanmics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg2+ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Membrane mobility agents: Alteration of human red blood cell membrane properties

Brief incubation of human red cells with the membrane mobility agent, 2-(2-methoxy)-ethoxyethyl 8-(2-n-octylcyclopropyl)-octanoate (A₂C), produces stomatocytes (with invaginations) along with a decrease in osmotic fragility, an altered distribution in a two-phase dextran-polyethylene glycol-water system, and increased permeability to the GSH oxidant, the diazene derivative, 1,2-diazenedicarboxylic acid bis(N′-methylpiperazide). These changes are consistent with what would be expected for agents which increase membrane lipid disorder. Membrane mobility agents provide a very convenient means for altering membrane properties and should be useful for studies on both normal and abnormal red cells.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Thermoelasticity of red blood cell membrane.

The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol.     

Viscoelastic properties of the human red blood cell membrane. II. Area and volume of individual red cells entering a micropipette.

Previous work demonstrated that human red cells can be drawn into cylindrical glass micropipettes of internal diameter approximately 2.0 mum without lysing. For pipettes of less than approximately 2.9 mum inside diameter, the red cell must become less spherical, that is, reduce its volume-to-area ratio. In this work measurements were made from 16-mm film records that allowed the determination of the cellular area and volume of individual erythrocytes as they were drawn into a 2.0-mum pipette with negative pressures. The results showed that the total surface area of the membrane remains constant and that the cell endures the passage into the pipette by losing volume. The volume loss was interpreted to be due to cell water and solute loss when the membrane is under stress. The loss of cell volume, rather than the stretching of the membrane, adds confirmation that although it is very deformable, the membrane is very resistant to two-dimensional strain.     

The human blood platelet: its derivation from the red blood cell. A morphologic study.

The blood platelet arises from the interior of the red blood cell when blood is either damaged or disturbed. The platelet body may be seen to form from an amorphous granular mass to a definite granular platelet body when blood is prevented from coagulating by the use of a retardant solution.     

Effect of hydroperoxides on red blood cell membrane mechanical properties

We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu.     

Kinetics of bicarbonate-chloride exchange across the human red blood cell membrane

The kinetics of bicarbonate-chloride exchange across the human red cell membrane was studied by following the time course of extracellular pH in a stopped-flow rapid-reaction apparatus during transfer of H+ into the cell by the CO2 hydration-dehydration cycle, under conditions where the rate of the process was determined by HCO3--Cl- exchange flux across the membrane. The flux of bicarbonate increased linearly with [HCO3-] gradient from 0.6 to 20 mM across the red cell membrane at both 37 degrees C and 2 degrees C, and decreased as transmembrane potential was increased by decreasing extracellular [Cl-]. An Arrhenius plot of the rate constants for the exchange indicates that the Q10 is strongly dependent on temperature, being about 1.7 between 24 degrees C and 42 degrees C and about 7 between 2 degrees C and 12 degrees C. These data agree well with the published values for Q10 of 1.2 between 24 degrees C and 40 degrees C and of 8 between 0 degrees C and 10 degrees C. The results suggest that different processes may determine the rate of HCO3- -Cl- exchange at low vs. physiological temperatures, and that the functional (and/or structural) properties of the red cell membrane vary markedly with temperature.     

Spectrin properties and the elasticity of the red blood cell membrane skeleton

Two models of spectrin elasticity are developed and compared to experimental measurements of the red blood cell (RBC) membrane shear modulus through the use of an elastic finite element model of the RBC membrane skeleton. The two molecular models of spectrin are: (i) An entropic spring model of spectrin as a flexible chain. This is a model proposed by several previous authors. (ii) An elastic model of a helical coiled-coil which expands by increasing helical pitch. In previous papers, we have computed the relationship between the stiffness of a single spectrin molecule (K) and the shear modulus of a network (μ), and have shown that this behavior is strongly dependent upon network topology. For realistic network models of the RBC membrane skeleton, we equate μ to micropipette measurements of RBCs and predict K for spectrin that is consistent with the coiled-coil molecular model. The value of spectrin stiffness derived from the entropic molecular model would need to be at least 30 times greater to match the experimental results. Thus, the conclusion of this study is that a helical coiled-coil model for spectrin is more realistic than a purely entropic model.     

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.     

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.     

Non-selective voltage-activated cation channel in the human red blood cell membrane

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.     

Elastic properties of the red blood cell membrane that determine echinocyte deformability

The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (A₀, the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, A₀ is modified and the corresponding discocyte–echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference A₀ is modified.     

The stress-free shape of the red blood cell membrane.

The two main proposals found in the literature for the stress-free shape of the red cell membrane are (a) the bioconcave shape and (b) the sphere of the same surface area. These possibilities are evaluated in this paper using theoretical modeling of equilibrium membrane shapes according to Zarda et al. (1977. J. Biomech. 10:211-221) and by comparison to experiments on red cells whose membrane shear modulus has been increased by treatment with diamide. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. To account for this discrepancy we propose that either the shear modulus of the native membrane is dependent on the membrane strain or that the bending stiffness is higher than estimated by Evans (1980. Biophys. J. 30:265-286). These studies suggest that the bioconcave disk is the more likely possibility for the stress-free shape.     

Interaction of hemoglobin with the red blood cell membrane. A saturation transfer electron paramagnetic resonance study

Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably slowed down when it is bound to the erythrocyte membrane. The correlation time of rotation, tau c, is found to be 8 . 10(-6) s as compared with 2 . 10(-8) s when the hemoglobin molecule is in solution. The same values are obtained whether the protein is associated with its high- or low-affinity binding sites. They depend on the viscosity of the solution. The high-affinity sites are presumably located on the segment of the band 3 protein which extends into the cytoplasm and which links through ankyrin, the spectrin-actin cytoskeleton to the membrane. When band 3 is cross-linked into a dimer after reaction with the copper-ortho-phenanthroline chelate, the correlation time of rotation of spin-labelled hemoglobin is unchanged. It is also independent of the presence of the spectrin-actin network and ankyrin. These results show tha the movements of hemoglobin bound by ionic linkage to different part (protein or phospholipid) of the cytoplasmic surface of the membrane are similarly highly restricted by some potential or energetic barrier. They give also evidence for independent movements and flexibility in the assembly of the macromolecules which link the spectrin-actin cytoskeleton to the erythrocyte membrane.