Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 μm in diameter.     

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.     

Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Excretion of superoxide by phagocytes measured with cytochrome c entrapped in resealed erythrocyte ghosts

Resealed erythrocyte membranes (ghosts) filled with (Fe3+)cytochrome c were used as an assay system to measure the release of superoxide (O-2) from human phagocytes into the incubation medium. Neutrophils, activated by either opsonized zymosan particles or the soluble stimulus phorbol myristate acetate, released O-2, which subsequently entered the ghosts and reduced (Fe3+)cytochrome c. This reaction was dependent on the time of incubation, the concentration of neutrophils, the concentration of stimulus, and the concentration of ghosts. The reaction was completely inhibited by superoxide dismutase and by 4,4'-diisothiocyano-2,2'-disulfonic acid, a specific blocker of anion channels in membranes. The reduction of (Fe3+)cytochrome c free in solution was about four times as fast as the reduction of (Fe3+)cytochrome c in the ghosts. Human eosinophils stimulated by phorbol myristate acetate reacted similarly to human neutrophils; the rate of O-2 production/cell was about twice as high for eosinophils as for neutrophils. In contrast, eosinophils stimulated with opsonized zymosan particles only reduced (Fe3+)cytochrome c free in solution, but not (Fe3+)cytochrome c in ghosts. This lack of reaction was not due to production of an inhibitor or below threshold generation of O-2 for the ghost assay. These results indicate: 1) activated human neutrophils and eosinophils can release O-2 or a similar product into the incubation medium; and 2) reduction of (Fe3+)cytochrome c free in solution is no proof for O-2 excretion by phagocytes.     

General and transport properties of hypotonic and isotonic preparations of resealed erythrocyte ghosts

Summary Resealed human erythrocyte ghosts are regarded as valuable tools for the study of membrane properties. In order to investigate to what extent preparation procedures affect the yield of ghosts, their general properties, and their permeability, ghosts prepared by lysis at low (hypotonic media) and high (isotonic media) ionic strength were compared with each other and with native erythrocytes. For isotonic lysis, cells were either subjected to dielectric breakdown or suspended in isotonic NH₄Cl solutions. In spite of very different characteristics of the lysis and the resealing process in the three types of preparations, the resulting ghosts do not differ in a number of features except for somewhat varying yields and for properties resulting from the mode of lysis.Specific transport properties, as characterized by the mediated fluxes ofm-erythritol,l-arabinose,l-lactate, and sulfate, proved to be unaltered with a few unsystematic exceptions. The simple nonmediated fluxes of all these permeants, as measured in the presence of inhibitors, however, were enhanced between 1.5- and 4-fold, indicating a somewhat increased ground permeability (of the lipid domain) in all ghost membranes.     

Amino acid transport by resealed ghosts from pigeon erythrocytes.

Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed.     

Neutralization of diphtheria toxin in living cells by microinjection of antifragment A contained within resealed erythrocyte ghosts

When human erythrocytes suspended in phosphate-buffered saline (PBS) containing lgG were first dialyzed against a hypotonic solution and then dialyzed against PBS, lgG molecules were entrapped within resealed erythrocyte ghosts. The concentration of lgG inside the ghosts was about 33% of its concentration in the dialysis bag. With the aid of HVJ (Sendai virus), ghosts containing rabbit lgG antibody against fragment A of diphtheria toxin were fused with toxin-sensitive FL cells. The fused FL recipients were found to be resistant to the action of diphtheria toxin. Clones derived from the resistant recipient cells, however, became sensitive to the toxin again. Antifragment A neutralized the enzymic activity of isolated fragment A in vitro, but did not protect FL cells or rabbit skin against the complete toxin.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

Intracellular gas supersaturation tolerances of erythrocytes and resealed ghosts.

Intact mammalian, avian, and amphibian erythrocytes were saturated with up to 300 atm nitrogen or argon gas and rapidly decompressed. Despite the profuse nucleation of gas bubbles in the suspending fluid, no evidence of intracellular gas bubble nucleation was found; all or most of the cells remained intact and little or no hemoglobin escaped. Internal bubbles were similarly absent from resealed ghosts of human erythrocytes as shown by lack of disintegration and by retention of an entrapped fluorescent compound. The absence of bubbles may indicate that much of the internal water does not have the same nucleation properties as external water.     

Prevention of nonspecific lysis in liposomal and erythrocyte immunoassay systems by small lipid vesicles and erythrocyte ghosts

Large unilamellar liposomes prepared by the reverse-phase evaporation method (REVs) were made immunoreactive by incorporating dinitrophenylaminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) or 8-(3-carboxypropyl)-theophylline-dipalmitoylphosphatidylethanolamine (Th-DPPE) into the phospholipid bilayer. Specific lysis in the presence of anti-DNP-BSA and goat anti-theophylline serum respectively, was induced by adding guinea pig serum as source for complement to these liposomes. However, specific lysis was found to be compromised by high levels of nonspecific lysis as monitored by the release of the fluorescent aqueous-space marker 6-carboxyfluorescein. Nonspecific lysis could be prevented without affecting specific lysis by pretreatment of complement or incubation of the reaction mixture with small unilamellar liposomes (SUVs). SUVs of various lipid compositions produced the desired effect; however, when the fraction of negative charge in the SUVs was increased to 30 mol%, specific lysis was inhibited as well. In a similar assay system consisting of hemolysin-sensitized sheep red blood cells it was also found that nonspecific lysis could be inhibited by addition of erythrocyte ghosts to the incubation medium, although specific lysis was somewhat depressed. However, SUVs or REVs of a composition similar to sheep erythrocytes were ineffective indicating a more selective nature of complement-mediated immunoreaction with erythrocyte membranes than with synthetic bilayer membranes.     

A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.     

The spontaneous activation of a potassium channel during the preparation of resealed human erythrocyte ghosts

Resealed erythrocyte ghosts prepared under conditions which deplete the cell of its endogenous chelators and metabolites are found to be selectively permeable to potassium. The net efflux of potassium is stimulated by low concentrations of external potassium and can be inhibited by oligomycin. The effect is not expressed when resealed ghosts are formed by hemolysis in the presence of chelators or magnesium. The spontaneously activated pathway is actually the calcium-activated potassium channel, first discovered by Gardos in 1958. In the intact cell, the combined actions of the calcium pump and endogenous chelators maintain the calcium concentration below the threshold for activation. Current observations indicate that the channel is spontaneously activated by traces of calcium originating from the cell itself or from the unavoidable background of calcium found in the media. The channel in ghosts depleted of endogenous chelators exhibits its high affinity for calcium. Channel activation occurs during hemolysis and persists throughout subsequent washings.     

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.     

Influence of the intracellular and extracellular cation concentration on monovalent cation efflux of resealed human erythrocyte ghosts

Tracer efflux measurements (⁸⁶Rb⁺ and²NaNa⁺) were performed on resealed human erythrocyte ghosts at different intra- and extracellular NaCI concentrations. Using a modified Goldman equation the observed alterations of the rate constants could be explained by taking into account the transmembrane and surface potentials, at constant permeability coefficient . These results emphasize the importance of membrane surface potentials in triggering ion transport across biological membranes.     

Organic-acid transport in resealed haemoglobin-containing human erythrocyte ''ghosts''.

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte 'ghosts' prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte 'ghosts' retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte 'ghosts' from patients with hyperoxaluria was also studied.     

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.