Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release

Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15°C or higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 μM for Ca²⁺ and 200 μM for Mg²⁺. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca²⁺, loss of spectrin does not occur. These results suggest that an intact spectrin network is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.     

Resealing to small solutes of white erythrocyte membranes after incubation with EDTA, Ca2+, salt, sucrose, phospholipase C

White, stable erythrocyte ghosts can recover their impermeability to small solutes after storage for several days in low-ionic-strength phosphate buffers at 0 °C. The accessibility, to their substrates, of the inner surface enzymes, glyceraldehyde-3-phosphate dehydrogenase, (G3PD), and NADH cytochrome c oxidoreductase, was used to assess resealing. The data from the two enzymes were confirmatory. None of the conditions used to investigate resealing altered the activity of the outer surface enzyme, acetylcholinesterase. Using G3PD activity, ghosts (freshly prepared by gentle stepwise hemolysis in hypotonic phosphate buffers and stored in 11 mm phosphate buffer, pH 7.4) were shown to be slightly sealed (33%). Incubation at 37 °C in the storage buffer with or without EDTA did not alter their permeability. Ionic strength rather than osmotic pressure appears to influence the sealing process since salt (286 mosm) elicited 91% sealing whereas sucrose (278 mosm) had little effect. Calcium in trace amounts caused resealing to 80%. Phospholipase C (C. welchii) completely abolished Ca²⁺-induced resealing. The data were highly reproducible although these ghosts were found to contain only 10 to 20% of the G3PD activity of the leaky ghosts prepared by shock hemolysis in 5 mm phosphate buffer, pH 8.0. The response to the resealing agents was similar regardless of the level of G3PD present. Neither calcium nor ETDA altered the chemical composition (sialic acid, cholesterol, phospholipid) of the membranes. The small amount (5%) of nonspecific loosely bound protein lost during incubation, could not be attributed to any of the test agents. The results suggest that calcium induced the recovery of impermeability by altering the association, distribution, and/or conformation of the proteins and phospholipids within the membrane.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Unmasking of a potassium leak in resealed human red blood cell ghosts.

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0 degrees C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. II. Formation of membrane leaks in ghost membranes after limited proteolysis of skeletal proteins by trypsin.

Limited proteolysis of human erythrocyte ghost membranes by low levels of trypsin (10-240 ng/ml) added bilaterally at 0 degrees C together with the proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF) before resealing at 37 degrees C leads to a graded digestion of spectrin and ankyrin and the disappearance of band 4.1 protein, while band 3 is cleaved only to a very low extent. These alterations are accompanied by an increase of membrane permeability of the resealed ghosts to hydrophilic nonelectrolytes (erythritol to sucrose), taken to reflect impaired resealing. Moreover, the membrane begins to vesiculate. Shedding of vesicles during the efflux measurements can not be responsible for the increased release of test solutes, since the ghosts do not loose hemoglobin and discriminate the nonelectrolytes according to their size. Moreover, the vesiculation site itself does not seem to act as the leak site, since ghosts prepared from erythrocytes pretreated with a carbodiimide which induces membrane rigidification still exhibit a pronounced protein degradation and vesiculation while the permeability enhancement induced by trypsination is markedly suppressed. The trypsin-induced leak has the properties of an aqueous pore as indicated, besides size selectivity, by its inhibition by phloretin and the very low activation energy. In analogy with concepts developed in the preceding paper (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)) the impaired resealing after limited proteolysis is assumed to be related to a perturbation of interactions of membrane skeletal elements with themselves and/or with the bilayer domain constituting the permeability barrier.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Sugar transport in reversibly hemolyzed avian erythrocytes

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37°C, 88% of the ghosts regained their permeability barrier to l-glucose. In these ghosts, the carrier-mediated rate of entry of $\text{3-O-}\text{methylglucose}$ was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : II. Antispectrin-Induced Transmembrane Aggregation of the Binding Sites for Positively Charged Colloidal Particles

The effects of affinity-purified antispectrin γ-globulins on the topographic distribution of anionic residues on human erythrocytes membranes was investigated using collo ida iron hydroxide labeling of mounted, fixed, ghost membranes. Antispectrin γ-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37°C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (<0.05 mg/ml) or high (>5–10 mg/ml concentrations of sequestered antispectrin was dispersed, but the distribution at intermediate concentrations (0.1–5 mg/ml) was highly aggregated. The aggregation of colloidal iron hydroxide binding sites was time and temperature dependent and required the sequestering of cross-linking antibodies (antispectrin Fab could not substitute for γ-globulin antibodies) inside the ghosts. Prior glutaraldehyde fixation or fixation at the time of hemolysis in antispectrin solutions prevented the antispectrin-induced colloidal iron site aggregation. The antispectrin reacted exclusively at the inner ghost membrane surface and the colloidal iron hydroxide bound to N-acetylneuraminic acid residues on the outer membrane surface which are overwhelming on the sialoglycoprotein glycophorin. These results were interpreted as evidence for a structural transmembrane linkage between the inner surface peripheral protein spectrin and the integral membrane component glycophorin.     

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Summary Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37°C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37° for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.     

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: Effects of lysing and resealing conditions

Summary The effects of various conditions in lysing and resealing the red cell membrane on the degree of ghost deformation and orientation in flow are investigated using the flow EPR and spin-label method. The relatively low deformability of the standard ghost, which is lysed and resealed, respectively, in hypotonic and isotonic NaCl-Tris buffer, is markedly enhanced by the presence of Mg-ATP, chlorpromazine, or Ca²⁺ ion during resealing. The effect is concentration dependent, and there is an optimal level for each treatment. Chlorpromazine and Ca²⁺ are also effective when added to the resealed ghosts. Mg²⁺ ion shows an opposite effect reducing the ghost deformability in flow at all concentrations. An isotonic lysis in NH₄HCO₃ solution with less osmotic stress substantially raises ghost deformability above that of the standard ghosts. These results are interpreted on the basis of a misalignment between the bilayer leaflets that is probably brought about during hypotonic lysis and its recovery to the nearly normal bilayer state by the agents used during or after resealing. The novel finding of deformability enhancing effect of calcium is assumed to be caused by the electrostatic expansion of the inner layer relative to the outer leaflet. The explanations are supported by the resealed ghost shapes observed before and after the treatments; shape recovery from the monoconcave spheroid toward biconcave discoid is observed in most cases concomitantly with improvements of flow characteristics.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Osmotic hemolysis contrasted with freeze-thaw hemolysis

Red blood cell ghosts resulting from osmotic hemolysis in the presence of Mg ions (Mg ghosts) and ghosts resulting from slow freeze-thaw process (freeze-thaw ghosts) differ in many respects: (1) Mg ghosts spontaneously take on the disc shape immediately after hemolysis and resuspension in buffered salt solution; whereas freeze-thaw ghosts are spherical; (2) Mg ghosts appear to be less hemolyzed than freeze-thaw ghosts; (3) washed and cold-stored Mg ghosts contract or become biconcave discs when exposed to 30 μmoles of ATP/10⁹ ghosts at 37 °C; whereas freeze-thaw ghosts under similar conditions break up into microspheres and membrane filaments; (4) Mg ghosts become crenated discs and spheres when rehemolyzed and resuspended in buffered salt solution; whereas freeze-thaw ghosts tend to fragment; (5) the ATPase activity of Mg ghosts, particularly the nontransport ATPase activity, is considerably less than that of freeze-thaw ghosts.     

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.     

Permeability properties of erythrocyte ghosts

1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described.     

Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK _m value of 2.56 · 10^–4 m and an apparentV _max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface. Abbreviations: PLC, phospholipase C; PIPLC, phospholipase C acting selectively on phosphatidylinositol; NeuAc,N-acetylneuraminic acid; MU, 4-methylumbelliferone; PBS, Dulbecco's phosphate buffer saline solution. Gangliosides were coded according to Svennerholm [42] and the IUPAC-IUB recommendations [43].     

Stimulation of calcium-sodium exchange in dog red blood cells by hemolysis and resealing

Osmotic hemolysis and resealing greatly increase calcium influx in dog red blood cells. The resealed ghosts show a saturable calcium entry pathway with complex kinetics. As expected for a calcium-sodium exchanger, calcium uptake is stimulated by internal sodium and inhibited by external sodium. Compared to fresh, intact red cells the resealed ghost calcium-sodium exchanger is less responsive to quinidine and to alterations in medium tonicity. The differences in calcium uptake rate among cells from different donors are minimized in the ghost preparation. There are several ways to stimulate sodium-dependent calcium movements in these cells, of which hemolysis-resealing is the most potent. The results of these and previous studies suggest that dog red blood cells have a latent capacity for calcium-sodium exchange.     

Cold-induced hemolysis in a hypertonic milieu

Summary Suspension of human erythrocytes at 37° C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0° C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20° C with an increasing effect up to 45° C, and the second stage consisting of lowering the temperature below 15° C with increasing hemolysis down to 0° C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.