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.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.     

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.     

A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.     

Characterization of calcium binding to spectrins.

Calcium binding to brain and erythrocyte spectrins was studied at physiological ionic strength by a calcium overlay assay and aqueous two-phase partitioning. When the spectrins were immobilized on nylon membranes by slot blotting, the overlay assay showed that even though both spectrins bound 45Ca2+, the brain protein displayed much greater affinity for calcium ions than erythrocyte spectrin did. Since the observed binding was weaker than that displayed by calmodulin under similar conditions, the overlay assay results indicated that the binding must be weaker than 1 microM. The phase partition experiments showed that there are at least two sites for calcium on brain spectrin and that calcium binding to one of these sites is reduced significantly by magnesium ions. From the partition isotherm, the dissociation constants were estimated as 50 microM for the Mg(2+)-independent site and 150 microM for the Mg(2+)-dependent site. The phase partition results also showed that erythrocyte spectrin bound calcium ions at least 1 order of magnitude weaker. By examining calcium binding to slot-blotted synthetic peptides, we identified two binding sites in brain spectrin. One mapped to the second putative calcium binding site (EF-hand) in alpha-spectrin and the other to the 36 amino acid residue long insert in domain 11. In addition, a tryptic fragment derived from the C-terminal of erythrocyte alpha-spectrin, which contained the two postulated EF-hands, also bound calcium. These findings suggest that the calcium signal system may also involve direct binding of calcium to spectrin beside known calcium modulators such as calmodulin and calpain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.     

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.     

Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types

The properties of brain fodrin have been analyzed and compared with those of erythrocyte spectrin. Both proteins consist of high molecular weight polypeptide doublets on SDS polyacrylamide gels and in solution behave as very large asymmetric molecules. Both proteins show a characteristic increase in sedimentation coefficient in the presence of 20 mM KCl. Antibodies against the brain protein cross-react with erythrocyte spectrin and cross-react with similar high molecular weight doublet polypeptides in SDS polyacrylamide gels of other cell types and plasma membrane preparations. Both proteins bind actin. The brain protein and erythrocyte spectrin show specific and competitive binding to erythrocyte membranes and this binding is inhibited by antibodies against erythrocyte ankyrin. Several of these properties distinguish these proteins from the class of high molecular weight actin-binding proteins that includes filamin and macrophage actin-binding protein. We conclude that together with erythrocyte spectrin, the brain protein and equivalent, immunologically related proteins in other cell types belong to a single class of proteins with the common function of attachment of actin to plasma membranes. Based on the structural and functional similarities, the name spectrin would seem appropriate for this whole class of proteins.     

Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells

In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537). The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of approximately 1 ankyrin to 4 Na,K-ATPase's, with a kD of 2.6 microM. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) 125I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of approximately 240 kD that is associated with both erythrocyte and kidney membranes that binds 125I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.     

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.     

Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.     

Influence of calmodulin on the human red cell membrane skeleton

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

Immunochemical characterization of interactions between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins

Autologous immunoglobulin G present during electrophoresis of human erythrocyte membrane proteins influenced the electrophoretic mobility of some of the proteins. Different types of non-ionic detergents were used for solubilization of the membranes and together with experiments using dimyristoylphosphatidylcholine-derived erythrocyte membrane vesicles this indicated that IgG binds to spectrin, ankyrin, and band 3 protein. The binding was independent on proteolysis and not due to unspecific protein-protein interactions. Immunoblotting experiments also showed binding to polypeptide bands in the spectrin and ankyrin regions and demonstrated the presence of erythrocyte-associated IgG. The reactivity may be due to natural autoantibodies involved in the clearance of cellular debris in vivo. Whether the observations are of relevance for the putative immune-mediated clearance of old erythrocytes from the circulation remains to be established.     

Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

A lymphoma plasma membrane-associated protein with ankyrin-like properties

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.