Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Phosphoinositide metabolism and the morphology of human erythrocytes

ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.     

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.     

Crenation and cupping of the red cell: A new theoretical approach. Part I. Crenation

Crenation can be thought of as a surface instability caused by intrinsic precurvature of the membrane. Mathematical modeling, on the presupposition that the red blood cell is a thin shell consisting of a connected (coupled) bilayer having uniformly distributed elastic properties shows that crenation can be initiated by negative precurvature, that is, intrinsic curvature having its concavity directed towards the outside of the cell. This is contrary to the currently accepted view which attributes the effect to positive precurvature of an unconnected bilayer. Crenation and the biconcave shape can coexist in the red cell. This suggests that the bilayer must be connected even when the cell is crenated because the biconcave shape could not otherwise be maintained. The progressive development of crenation to more advanced stages, such as the echinocyte type III and the spheroechinocyte can be accounted for if the outer layer of the membrane is stressed beyond the range where strain is proportional to stress. This is consistent with the extremely small radius of curvature at the tips of the crenations.Certain small variations in the uncrenated biconcave shape of the red cell can be interpreted mathematically as due either to negative intrinsic curvature or to shear resistance. Since, however, a small amount of negative precurvature has been shown to be capable of inducing crenation, it is unlikely to be the cause of the variations in the biconcave shape. These must therefore be due to shear resistance.In the light of this new approach, membrane molecular models based on the assumption that crenation is due to positive precurvature need reconsideration.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Spectrin phosphorylation and shape change of human erythrocyte ghosts

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.     

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry

The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.     

Asymmetric manipulation of the membrane lipid bilayer of intact human erythrocytes with phospholipase A, C, or D induces a change in cell shape

Changes in the membrane morphology and phospholipid content of human erythrocytes were determined after incubation of intact cells with each of various exogeneous phospholipases (PLases). PLase A2 from Naja naja or bee venom induced crenation of the cells in parallel with hydrolysis of the membrane phosphatidylcholine (PC). This crenated cell shape was reversed to a biconcave disc or cup-like form by a further treatment with lysophospholipase. In contrast, bacterial PLase C from Clostridium perfringens and Pseudomonas aureofaciens or fungal PLase D from Streptomyces chromofuscus induced invagination of the cells in parallel with hydrolysis of the PC. The action of the latter group of PLases on the membrane morphology was counteracted by PLase A2, and vice versa. Thus, participation of the membrane lipid bilayer in the induction of membrane conformational change and hence cell shape change was demonstrated.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Different arachidonate and palmitate binding capacities of the human red cell membrane

Human red cell membrane bindings of arachidonate and palmitate at pH 7.3 are investigated at temperatures between 0 and 38°C by equilibrating ghosts with the long-chain fatty acids bound to bovine serum albumin in molar ratios (v) within the physiological range (<1.7). Linearized relations of ghost uptakes and fatty acid monomer concentrations in buffer provide estimates of the binding capacities and corresponding equilibrium dissociation constants (K _dm ). The temperature-independent arachidonate binding capacity, 5.5 ± 0.5 nmol g^–1 packed ghosts, is approximately fivefold smaller than that of palmitate, 26.6 ± 2.0 nmol g^–1. While K _dm of arachidonate binding 5.1 ± 0.5 nm is temperature independent, K _dm of palmitate increases with temperature from 3.7 nm at 0°C to 12.7 nm at 38°C.The large difference in binding capacities suggests the presence of at least two different fatty acid binding domains in human red cell membranes.     

Mechanism of red blood cell acanthocytosis and echinocytosis in vivo

Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10 X 10(-5) M chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, concave disks by chlorpromazine or merely by a brief incubation at 37 degrees C in low ionic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from a patient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenation in vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronically abnormal in shape in vivo contain the information to become biconcave disks immediately upon simple chemical treatment in vitro.     

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.     

Mechanism of red blood cell acanthocytosis and echinocytosisin vivo

Summary Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10×10^–5 m chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, convave disks by chlorpromazine or merely by a brief incubation at 37°C in low inoic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from apatient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenationin vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronicallyabnormal in shapein vivo contain the information to become biconcave disks immediately upon simple chemical treatmentin vitro.     

Asymmetric binding of cytochrome b5 to the membrane of human erythrocyte ghosts

The intact, amphipatic form of cytochrome $\text{b}{}_5$ could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome $\text{b}{}_5$ to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.     

Separation of the lipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts

The membrane skeleton, a protein lattice at the internal side of the red cell membrane, is principally composed of spectrin, actin and proteins 4.1 and 4.9. We have examined negatively stained red cell ghosts and demonstrated, on an ultrastructural level, a separation of the lipid bilayer from the membrane skeleton during echinocytic transformation. The electron micrographs of discoidal red cell ghosts suspended in hypotonic buffer revealed a filamentous reticulum that uniformly laminated the entire submembrane region. transformation of the discoidal ghosts into echinocytic form, as induced by incubation in isotonic buffer, resulted in a disruption of skeletal continuity underlying the surface contour of the membrane spicule. The submembrane reticulum extended into the base and the neck of the spiny processes of the crenated ghosts but was absent at the tip of these projections. In addition, membrane vesicles without a submembrane reticulum were detected either attached to the tips of the spicules or released into the supernatant from the echinocytic ghosts. Protein analysis revealed that the released vesicles were enriched in bands 3, 4.1 and 7 and contained very little of the membrane skeletal proteins, spectrin and actin. The data indicate that during echinocyte formation, parts of the lipid bilayer physically separate from the membrane skeleton, leading to a formation of skeleton-poor lipid vesicles.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Summary ³H-labelled phloretin was shown to be bound reversibly by human erythrocyte and ghost membranes but not to penetrate across them in either direction. Kinetic parameters ofd-xylose andd-galactose transport in intact cells and in ghosts, as well as the inhibition by phloretin of these transports were found to be in fair agreement. By enclosing phloretin in ghosts, its inhibition of monosaccharide transport was found to be symmetrical and thus an equivalence of the outer and the inner membrane sides of the human erythrocyte was demonstrated.     

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.