The size of erythrocyte ghosts

The volume of resealed erythrocyte ghosts formed during hypotonic hemolysis of normal human erythrocytes was measured by means of a continuous mean corpuscular volume analyzer. The final volume of resealed ghosts was 140.6 ± 15.2 fl. Strong correlations exist between the volume of ghosts and the initial mean corpuscular volume and mean corpuscular hemoglobin of the erythrocyte, and between the enlargement ratio and the mean corpuscular volume or mean corpuscular hemoglobin of the erythrocyte.     

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.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.     

The effect of cyclic nucleotides and protein phosphorylation on the permeability of human erythrocyte ghosts to certain cations

Summary Preparations of human erythrocyte membranes have been made which are in the form of sealed vesicles and which behave as osmometers on suspension in solutions of simple inorganic salts. Using these preparations the permeability of the membranes to Na⁺, K⁺, Mg²⁺ and Ca²⁺ was measured. Cyclic AMP (but not cyclic GMP) increased the permeability of the membranes to Ca²⁺ with a half maximal effect at a concentration of 25µm but did not affect the permeability to the other ions tested. Phosphorylation of proteins in the erthrocyte membrane lowered the permeability to Ca²⁺ without affecting the permeability to the other ions tested and there was a good correlation between the time course of protein phosphorylation and decrease in Ca²⁺ permeability.It is postulated that the system through which cyclic AMP causes an initial rapid rise in Ca²⁺ permeability followed by increased phosphorylation of membrane proteins and reduced Ca²⁺ permeability may have a widespread occurrence in biological systems and serve to control the concentration of Ca²⁺ in the cytoplasm.     

Calorimetric measurements of the effect of 330-MHz radiofrequency radiation on human erythrocyte ghosts

Irreversible changes in the heat capacity of human erythrocyte ghost suspensions due to the effect of 330-MHz radiofrequency radiation (at a specific absorption rate of approximately 9 mW/g) were detected by the method of scanning differential microcalorimetry. Using the data obtained from the analysis of infrared spectra of air-dried films of erythrocyte membranes, it can be postulated that the observed microcalorimetric changes are connected with the local interaction of electromagnetic radiation with the channel-forming portion of band-3 protein.     

Human lymphocyte receptor movement induced by sheep erythrocyte binding: effect of temperature and neuraminidase treatment

The formation of sheep red blood cells (SRBC) rosettes by human lymphocytes was promoted by incubation at 4 °C and by treatment of the lymphocytes or SRBC by neuraminidase. On incubating the untreated SRBC rosettes at 37 °C, the rosettes dissociated by capping in which rings were converted into horseshoes and then caps. This capping was inhibited by incubation of the rosettes at 4 °C and partially by treatment of the cells with neuraminidase. During rosette formation, the proportion of caps decreased progressively during 4 °C incubation. This decrease of capping was also promoted by neuraminidase treatment. We concluded that the main reason why 4 °C and neuraminidase treatment facilitated rosette formation was by inhibition of capping.     

Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts.

A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.     

Rotational diffusion of band 3 in erythrocyte membranes. 1. Comparison of ghosts and intact cells.

The rotational diffusion of eosin-labeled 3 in human erythrocyte cells and hemoglobin-free ghosts at 37 degrees C has been studied in detail by polarized delayed luminescence. The time-resolved anisotropy with both cells and freshly prepared ghosts is similar, decaying with well-resolved rotational correlation times of 0.03, 0.2, and greater than or equal to 1 ms. Mild proteolytic removal of the water-soluble 41-kDa cytoplasmic domain of band 3 in ghosts results in a drastic increase in the fractional contributions of the two fastest depolarizing components. Our results, taken together with other data in the literature, imply that several classes of band 3 that differ greatly in mobility exist in ghosts and intact cells. The mobility of one class is hindered due to complexation with other membrane or cytoplasmic proteins mediated via the 41-kDa cytoplasmic domain. However, another class of band 3 molecules exists as homo-or heterooligomeric complexes larger than a dimer that are stabilized by hydrophobic interactions involving the intramembranal domain. Finally, the presence of the (previously undetected) 0.03-ms anisotropy component strongly suggests that a significant fraction of band 3 in both ghosts and intact cells is highly mobile and diffuses at the rate expected for a freely rotating dimer in the erythrocyte membrane.     

The control of adenylate cyclase by calcium in turkey erythrocyte ghosts.

The adenylate cyclase of turkey erythrocytes is inhibited by low concentrations of calcium. Calcium binds to the enzyme system so tightly that the enzyme can compete with ethylene glycol bis(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA) for the metal. The calcium binding site is shown to be distinct from the magnesium binding sites required for activity. Thus Ca2+ functions as a negative allosteric effector. Calcium decreases dramatically the V max of the catecholamine-stimulated activity without affecting the affinity for the hormone or for the substrate ATP. The cooperativity in the response toward Mg2+ dependence (Hill coefficient, nH equals 3) is also unaffected by Ca2+ where as the S0.5 (concentration yielding one-half V max) for Mg2+ is affected only slightly. The Ca2+ effect is cooperative (nH equals 2) and therefore brought about by a cluster of Ca2+ binding sites. Mn2+ can substitute for Mg2+ as the enzyme activator but the Mn2+-activated enzyme is no longer inhibited by Ca2+. The possible physiological significance of the Ca2+ effect is discussed.     

Effect of pulse electromagnetic radiation on erythrocyte ghosts

The pulse microwave radiation has been shown to increase the fluorescence intensity of 2-toluidinonaphthanene-6-sulfonate (2,6-TNS) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) built-in membranes of erythrocyte ghosts. In experiments with 2,6-TNS a frequency dependence of the effect of microwave radiation with maximum within the frequency range of 55-65 Hz has been found. It is suggested that the changes registered with fluorescent probes are induced by mechanical oscillations generated by the pulse microwave radiation.