Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.     

Alteration of alpha-spectrin ubiquitination due to age-dependent changes in the erythrocyte membrane.

Mammalian red blood cell alpha-spectrin is ubiquitinated in vitro and in vivo [Corsi, D., Galluzzi, L., Crinelli, R., Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935]. This process shows a cell age-dependent decrease, with senescent red blood cells having approximately one third of the amount of ubiquitinated alpha-spectrin found in young cells. In-vitro ubiquitination of alpha-spectrin was dependent on the source of the red cell membranes (those from older cells are less susceptible to ubiquitination than those from younger cells), on the source of ubiquitin-conjugating enzymes (those from older cells catalyze the process at a reduced rate compared to those from younger cells) and on the ubiquitin isopeptidase activity (which decreases during red cell ageing). However, once alpha-spectrin has been extracted from the membranes of young or old red blood cells, it is susceptible to ubiquitination to a similar extent regardless of source. This suggests that it is the membrane architecture, and not spectrin itself, that is responsible for the age-dependent decline in ubiquitination. Furthermore, spectrin oligomers, tetramers and dimers are also equally susceptible to ubiquitination. As spectrin ubiquitination occurs on domains alphaIII and alphaV of alpha-spectrin, and domain alphaV contains the nucleation site for the association of the alpha- and beta-spectrin chains, alterations in ubiquitination during red cell ageing could affect the stability and deformability of the erythrocyte membrane.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

A spectrin-like protein in retinal rod outer segments

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.     

Oxidative erythrocyte membrane damage in hereditary spherocytosis.

The occurrence, in Hereditary Spherocytosis, of an oxidative damage to red blood cell membranes was studied by "in vitro" treatment of the erythrocytes with tert-butylhydroperoxide, methylene blue, or phenylhydrazine. Spherocytes were found to be more sensitive than normal erythrocytes to the action of these drugs. Tert-butylhydroperoxide caused a more intense lipid peroxidation as well as more extensive membrane protein alterations, namely spectrin degradation, formation of high molecular weight aggregates, and globin binding to the membrane. Marked spectrin degradation was also induced by methylene blue and by phenylhydrazine, which differed from each other for their effects on the generation of membrane-bound globin and of intermediate proteolysis products. Spectrin appeared therefore to be, in Hereditary Spherocytosis, a highly sensitive target to oxidative stress, a phenomenon which may, also "in vivo", increase the rate of spectrin loss thus enhancing erythrocyte fragility.     

Intramolecular dynamics of human erythrocyte membrane proteins upon modification of spectrin by tryptophan phosphorescence

The slow (millisecond) protein internal dynamics of isolated human erythrocyte membranes in suspension without treatment, after deleting 95% of spectrin, after spectrin thermal denaturation upon acidification of medium in the pH range 6.0-4.0, and spectrin extracted in solution from membranes has been studied by room-temperature tryptophan phosphorescence. It has been established that integral proteins and spectrin differ in structural and dynamic state. Millisecond movements of structural elements of integral proteins are more restricted compared with those of spectrin. The removal of spectrin from the membrane led to an increase in slow fluctuations of integral protein structure. This indicates that spectrin participates in the control of the structural and dynamic state of erythrocyte membrane proteins. As medium was acidified in the pH range 6.0-4.0, the protein slow internal dynamics of membranes in native state decreased, which was explained by spectrin pH aggregation. After thermal denaturation of spectrin, no pH-induced increase of membrane protein structure rigidity was observed.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.     

Effects of inherited membrane abnormalities on the viscoelastic properties of erythrocyte membrane.

Several workers have identified molecular abnormalities associated with inherited blood disorders. The present work examines how these alterations in molecular structure affect the viscoelastic properties of the red blood cell membrane. Changes in the membrane shear modulus, the membrane viscosity, and the apparent membrane bending stiffness were observed in cells of eight patients having a variety of disorders: Two had reductions in the number of high-affinity ankyrin binding sites, two had abnormalities associated with the protein band 4.1, and six were known to be deficient in spectrin. The data suggest that the membrane shear modulus is proportional to the density of spectrin on the membrane and support the view that spectrin is primarily responsible for membrane shear elasticity. Although membranes having abnormalities associated with the function of ankyrin or band 4.1 exhibited reduced elasticity, the degree of mechanical dysfunction was quantitatively inconsistent with the extent of the molecular abnormality. This indicates that these skeletal components do not play a primary role in determining membrane shear elasticity. The membrane viscosity was reduced in seven of the eight patients studied. The reduction in viscosity was usually greater than the reduction in shear modulus, but the degree of reduction in viscosity was variable and did not correlate well with the degree of molecular abnormality.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Cross-linking of erythrocyte membrane proteins by periodate and intramembrane particle distribution.

Treatment of isolated human erythrocyte membranes at pH 7.4 with 0.1-0.5 mM-sodium periodate specifically cross-linked some of the spectrin polypeptides. Treatment with 2 mM-periodate resulted in complete cross-linking of spectrin and partial cross-linking of other polypeptides. The latter treatment also caused aggregation of the intramembrane particles made visible by freeze-fracturing. When membranes that had been treated with 2 mM-periodate were depleted of spectrin by treatment with 0.1 mM-EDTA, extensive aggregation of the intramembrane particles occurred.     

Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin

Ternary complex formation between the major human erythrocyte membrane skeletal proteins spectrin, protein 4.1, and actin was quantified by measuring cosedimentation of spectrin and band 4.1 with F-actin. Complex formation was dependent upon the concentration of spectrin and band 4.1, each of which promoted the binding of the other to F-actin. Simultaneous measurement of the concentrations of spectrin and band 4.1 in the sedimentable complex showed that a single molecule of band 4.1 was sufficient to promote the binding of a spectrin dimer to F-actin. However, the molar ratio of band 4.1/spectrin in the complex was not fixed, ranging from approximately 0.6 to 2.2 as the relative concentration of added spectrin to band 4.1 was decreased. A mole ratio of 0.6 band 4.1/spectrin suggests that a single molecule of band 4.1 can promote the binding of more than one spectrin dimer to an actin filament. Saturation binding studies showed that in the presence of band 4.1 every actin monomer in a filament could bind at least one molecule of spectrin, yielding ternary complexes with spectrin/actin mole ratios as high as 1.4. Electron microscopy of such complexes showed them to consist of actin filaments heavily decorated with spectrin dimers. Ternary complex formation was not affected by alteration in Mg2+ or Ca2+ concentration but was markedly inhibited by KCl above 100 mM and nearly abolished by 10 mM 2,3-diphosphoglycerate or 10 mM adenosine 5'-triphosphate. Our data are used to refine the molecular model of the red cell membrane skeleton.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

Brain protein 4.1 subtypes: a working hypothesis

In a companion review¹ we discussed the data supporting the conclusion that at least two subtypes of spectrin exist in mammalian brain. One form is found in the cell bodies, dendrites, and post-synaptic terminals of neurons (brain spectrin(240/235E)) and the other subtype is located in the axons and presynaptic terminals (brain spectrin(240/235)). Our recent understanding of brain spectrin subtype localization suggests a possible explanation for a conundrum concerning brain 4.1 localization. Amelin, an immunoreactive analogue of red blood cell (rbc) cytoskeletal protein 4.1, is localized in neuronal cell bodies and dendrites when brain sections are stained with antibody against rbc protein 4.1. However, it has recently been suggested that synapsin I, a neuron-specific phosphoprotein associated with the cytoplasmic surface of small synaptic vesicles, is related to erythrocyte 4.1. In this review we hypothesize that there are at least two forms of brain 4.1: a cell body/dendritic form (amelin) which is detected with rbc protein 4.1 antibody, and a unique form found exclusively in the presynaptic terminal (synapsin I). The binding of synapsin I to brain spectrin(240/235), and its ability to stimulate the spectrin/F-actin interaction in a phosphorylation-dependent manner suggests a model for the regulation of synaptic transmission mediated by the neuronal cytoskeleton.     

Purification and properties of human erythrocyte band 4.2. Association with the cytoplasmic domain of band 3

We have purified the human erythrocyte membrane protein band 4.2 to greater than 85% homogeneity. The protein was extracted from spectrin-actin-depleted inside-out vesicles in a pH 11 medium and purified by gel filtration in the presence of 1 M KI. The purified protein was heterogeneous and had an average S20,w of 5.5 and an average Stokes radius of 82 A. By electron microscopy, the protein appeared heterogeneous in size and shape, having a diameter ranging from 80 to 150 A. The protein bound saturably to band 4.2-depleted red cell inside-out vesicles, and the binding exhibited a concave Scatchard plot. Binding was reduced greater than 90% by proteolytic digestion of membranes. Digestion studies suggested that there are two classes of binding sites for band 4.2 on the cytoplasmic aspect of red cell membranes, one of which is likely to be band 3. The purified 43-kDa cytoplasmic domain of band 3 competed for band 4.2 binding to red cell membranes and could completely abolish binding when added at a concentration of greater than 200 micrograms/ml. The purification of band 4.2 and the characterization of its association with red cell membranes should facilitate the discovery of the function of this major red cell membrane protein.