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.     

Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.     

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.     

Cystic fibrosis: II. Altered microviscosity of erythrocyte membrane

Erythrocyte membrane fluidity alterations in cystic fibrosis are described. The relative flexibility of the membrane was studied using lipid spin label, i.e. methyl-5-doxylpalmitate (M5DP), and pyrene as a fluorescence probe. It was found that there was a decrease of membrane fluidity in the hydrophobic midzone of the membrane, probed by pyrene, as well as at the hydrophilic surface region, probed by M5DP.     

A method for measuring linearly viscoelastic properties of human tympanic membrane using nanoindentation

A viscoelastic nanoindentation technique was developed to measure both in-plane and through-thickness viscoelastic properties of human tympanic membrane (TM). For measurement of in-plane Young's relaxation modulus, the TM sample was clamped on a circular hole and a nanoindenter tip was used to apply a concentrated force at the center of the TM sample. In this setup, the resistance to nanoindentation displacement can be considered due primarily to the in-plane stiffness. The load-displacement curve obtained was used along with finite element analysis to determine the in-plane viscoelastic properties of TM. For measurements of Young's relaxation modulus in the through-thickness (out-of-plane) direction, the TM sample was placed on a relatively rigid solid substrate and nanoindentation was made on the sample surface. In this latter setup, the resistance to nanoindentation displacement arises primarily due to out-of-plane stiffness. The load-displacement curve obtained in this manner was used to determine the out-of-plane relaxation modulus using the method appropriate for viscoelastic materials. From our sample tests, we obtained the steady-state values for in-plane moduli as approximately 17.4 MPa and approximately 19.0 MPa for posterior and anterior portions of TM samples, respectively, and the value for through-thickness modulus as approximately 6.0 MPa for both posterior and anterior TM samples. Using this technique, the local out-of-plane viscoelastic modulus can be determined for different locations over the entire TM, and the in-plane properties can be determined for different quadrants of the TM.     

A new spin label method for the measurement of erythrocyte internal microviscosity

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.     

Cyclic AMP transport in human erythrocyte ghosts.

10(-5) M cyclic AMP has high permeability in human erythrocyte ghosts (p = 0.061-10(-6) cm.s-1). Saturation of influx and efflux occurs. Koizt = 4.43 mM. Voizt = 259.6 micron.min-1-Kiozt = 0.475 micron. Viozt = 28.3 micron.min-1 at 30 degrees C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cytochalasin B is an apparent competitive inhibitor of cyclic AMP exit. (Ki = 3.9.10(-7) M). Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Structural properties of DNO investigated with pyrene excimer formation.

Four diamino acid-Nalpha-substituted oligopeptide (DNO) oligomers substituted with pyrenyl as photophysical probes were synthesized. The excimer formation and ground-state association of the pyrenyl groups were investigated by means of absorption and steady-state fluorescence spectroscopy together with time-resolved fluorescence techniques. The photophysical parameters obtained from the different derivatives reflect the secondary structural properties of the DNO backbones.     

Insulin causes insulin-receptor internalization in human erythrocyte ghosts.

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.     

Study of the aggregation of membrane proteins of erythrocyte ghosts using SDS-PAAG electrophoresis

Using the method of electrophoresis in SDS-PAAG the authors showed a diminution of proteins of bands I + II (spectrins) and III (major integral protein) after irradiation of erythrocyte ghosts with doses of 50 to 1000 Gy. We failed to ascertain that radiation-induced lipid peroxidation is involved into membrane protein aggregation. Among the radiolysis products, OH-radicals were shown to contribute markedly to the radiation effect observed.     

Effect of hemoglobin A and S on human erythrocyte ghosts

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

A new method for measuring binding of labeled ligands to membrane receptors.

Two different automated procedures for protein microdetermination by the Lowry method have been devised to analyze samples with high and low concentrations of nonionic detergents. Autoanalytical monitoring of the protein concentration of column effluents has been achieved by substituting 50 mm Tricine for 50 mm Tris in an eluting buffer containing 0.05% Poly-Tergent.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.