ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

ESR studies of the erythrocyte membrane skeletal protein network: Influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bis-phosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P < 0.002 and P < 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding sites is observed (P < 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P < 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.     

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.     

p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.     

Triton shells of intact erythrocytes

About 40% of human erythrocyte membrane protein is resistant to solubilization in 0.5% Triton X-114. These components comprise a structure called a Triton shell roughly similar in size and shape to the original erythrocyte and thus constitute a cytoskeleton. With increasing concentrations of Triton the lipid content of the Triton shell decreases dramatically, whereas the majority of the protein components remain constant. Exceptions to this rule include proteins contained in band 3, the presumed anion channel, and in band 4 which decrease with increasing Triton concentration. The Triton-insoluble complex includes spectrin (bands 1 and 2), actin (band 5), and bands 3′ and 7. Component 3′ has an apparent molecular weight of 88,000 daltons as does 3; but unlike 3, it is insensitive to protease treatment of the intact cell, has a low extinction coefficient at 280 nm, and is solubilized from the shells in alkaline water solutions. Component 7 also has a low extinction coefficient at 280 nm. Spectrin alone is solubilized from the Triton shells in isotonic media. The solubilized spectrin contains no bound Triton and coelectrophoreses with spectrin eluted in hypotonic solutions from ghosts. Electron micrographs of fixed Triton shells stained with uranyl acetate show the presence of numerous filaments which appear beaded and are 80–120 Å in diameter. The filaments cannot be composed mainly of actin, but enough spectrin is present to form the filaments. Triton shells may provide an excellent source of material useful in the investigation of the erythrocyte cytoskeleton.     

Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation.

Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles.     

Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state

The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Spin-Labelling Studies of the Interaction of 9-Amino-1,2,3,4-Tetrahydroacridine (Tha), A Proposed Drug for the Treatment of Alzheimer's Disease,with Erythrocyte Membranes

ESR spin labels specific for skeletal proteins or cell-surface sialic acid have been used to monitor the interaction of 9-amino-1,2,3,4-tetrahydroacridine (THA) and its structural analogs with human eryth-rocyte membranes. The results suggest that THA significantly increases skeletal protein-protein interactions and may secondarily alter the physical state of the opposite side of the membrane. The fully aromatic analog of THA, 9-aminoacridine, showed even more pronounced effects on skeletal proteins than did THA. These results are discussed in relation to possible interaction sites of THA in erythrocyte ghosts and to potential mechanisms by which THA reportedly increases mental function of victims of Alzheimer's disease.     

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.     

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles.

Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts. Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregation of the particles. With virtually no spectrin present, the particles in these stripped ghosts could still be aggregated by manipulations with ionic strength and pH, or by the addition of calcium. Recombinant vesicles were made from a Triton X-100 extract and a mixture of phospholipids with a composition which resembled that of the inner monolayer of erythrocyte membrane. In these recombinants the same manipulations with ionic strength and pH and the addition of calcium caused a rearrangement of the particles, resulting in the appearance of particle-free areas. In recombinants prepared from a Triton X-100 extract and egg phosphatidylcholine the lateral distribution of the particles was not altered by these manipulations. It is concluded that in the erythrocyte membrane the intramembrane particles can be aggregated by effects of external agents on lipid components. In this light the role of spectrin in stabilizing the membrane by interactions with lipids in the inner monolayer is discussed.     

Alternations in lipid membrane fluidity and the physical state of cell-surface sialic acid in zinc-deficient rat erythrocyte ghosts

Erythrocyte ghosts, prepared from the blood of rats fed zinc-deficient diets, were evaluated for membrane fluidity and surface sialic acid properties using spin-labeled probes and electron spin resonance (ESR) spectroscopy. These physical parameters of the erythrocyte ghosts from the zinc-deficient group were compared to those for erythrocyte ghosts obtained from ad libitum and pair fed controls consuming zinc-adequate diets. As the animals became progressively zinc deficient, the erythrocyte ghost membranes became more fluid than those from the control groups. In addition, the apparent rotational correlation time of Tempamine spin probes on surface sialic acid residues was smaller for the zinc deficient group, indicative of an increased rotational mobility of the spin label. These results suggest that zinc deficiency can have pronounced effects on the physical state of membrane bilayer lipids and cell surface carbohydrates and supports the view that many of the pathological signs of zinc deficiency are due to a general membrane defect.     

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'- Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain on its interaction with cytoskeletal elements. In contrast, these barrier properties seem to be rather insensitive to perturbations of integral proteins.     

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts.Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregation of the particles. With virtually no spectrin present, the particles in these stripped ghosts could still be aggregated by manipulations with ionic strength and pH, or by the addition of calcium.Recombinant vesicles were made from a Triton X-100 extract and a mixture of phospholipids with a composition which resembled that of the inner monolayer of erythrocyte membrane. In these recombinants the same manipulations with ionic strength and pH and the addition of calcium caused a rearrangement of the particles, resulting in the appearance of particle-free areas. In recombinants prepared from a Trixon X-100 extract and egg phosphatidylcholine the lateral distribution of the particles was not altered by these manipulations.It is concluded that in the erythrocyte membrane the intramembrane particles can be aggregated by effects of external agents on lipid components. In this light the role of spectrin in stabilizing the membrane by interactions with lipids in the inner monolayer is discussed.     

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.     

A minor sialoglycoprotein of the human erythrocyte membrane

The sialoglycoprotein periodate fuchsin sulfite 2 has about 8% of the sialic acid contained in the sialoglycoproteins of the human erythrocyte membrane. This polypeptide appears to have an apparent monomeric molecular weight of 35,000, somewhat smaller than the monomer of the major sialoglycoprotein (periodate fuchsin sulfite 1) as judged by sodium dodecyl sulfate-polyacry lamide gel electrophoresis, and has frequently been confused with the monomer of the major sialoglycoprotein. Periodate fuchsin sulfite 2 is not labeled by the lactoperoxidase procedure in the intact cell, although it is accessible to neuraminidase and other hydrolases. On the other hand, this component can be labeled by lactoperoxidase on the cytoplasmic surface of open membranes or resealed ghosts. Thus, it is a trans membrane protein. Although most of the other transmembrane proteins of the human erythrocyte membrane are extracted from the membrane by 0.1% Triton X-100 in 7 mm phosphate buffer, pH 7.4, this component is not removed and may be a cytoskeletal component. Trypsin, chymotrypsin, and thermolysin peptides, as well as cyanogen bromide fragments, clearly indicate that the primary sequence of this polypeptide can be distinguished from dimeric or monomeric forms of the major sialoglycoprotein (periodate fuchsin sulfite 1).     

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium- dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans- marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.