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PubMed1997

Definition of a sequence unique in beta II spectrin required for its axon-specific interaction with fodaxin (A60).

Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon-specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of alpha I, beta I, and beta II polypeptides) but not erythrocyte spectrin (alpha I and beta I). Because alpha II and beta II spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain alpha chains. Bacterially expressed C-terminal segments 18-19 of beta II spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18-19 of the somatodendritic beta I sigma 2 spectrin showed no interaction with fodaxin. Within beta II, fodaxin binding activity was localized to residues 2,087-2,198, which are unique to beta II and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of beta II and beta I sigma 2 are candidates to mediate the isotype-specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of beta spectrin isoforms.

Animals↗
PubMed1998

A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles.

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the beta-spectrin gene family by homology searches of the GenBank databases and by 5' rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called betaIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. betaIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human betaIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human betaIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that betaIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and beta-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with betaIII spectrin. betaIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Animals↗
PubMed2001

beta III spectrin binds to the Arp1 subunit of dynactin.

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

Actins↗
PubMed1990

A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin.

Spectrins are a major component of the membrane skeleton in many cell types where they are thought to contribute to cell form and membrane organization. Diversity among spectrin isoforms, especially their beta subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD) that we term beta H. The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit but shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family such as alpha-spectrin and alpha-actinin.

Amino Acid Sequence↗
PubMed1995

Drosophila development requires spectrin network formation.

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.

Amino Acid Sequence↗
PubMed1996

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

Adult↗
PubMed1986

Occurrence of spectrin-like protein in Y-1 adrenal tumor cells.

With the aid of two monospecific antibodies raised in rabbits (antimouse erythrocyte spectrin and antimouse brain spectrin), the presence of a spectrin-like protein was demonstrated in mouse adrenal tumor (Y-1) cells. Y-1 cells contain two large polypeptides, with mol wt characteristic of nonerythroid spectrin alpha- and beta-subunits (240,000 and 235,000). When proteins from plasma membranes of Y-1 cells were electrophoretically transferred to a nitrocellulose membrane, two polypeptides with mol wt of 240,000 and 225,000 were specifically stained with antimouse erythrocyte (rbc) spectrin immunoglobulin G (IgG). The rbc spectrin antibody was used to immunoprecipitate Y-1 spectrin from a neutral detergent (physiological ionic strength) cell extract. The 240,000 (alpha)- and 235,000 (beta)-dalton polypeptides were immunoprecipitated in a 1:1 molar ratio, despite the fact that the antibody recognizes only the alpha-subunit. Two-dimensional chymotryptic peptide-mapping analysis indicated that the 240,000- and 235,000-dalton subunits of Y-1 adrenal tumor spectrin are structurally unique and share limited homology with mouse rbc spectrin alpha- and beta-subunits, but are nearly identical to the mouse brain spectrin 240,000-dalton alpha-subunit and 235,000-dalton beta-subunit. Indirect immunofluorescence with anti-rbc or antibrain spectrin IgG and goat antirabbit IgG conjugated with rhodamine demonstrated intense staining at the plasma membrane and throughout the cytoplasm of Y-1 cells, with little staining within the nucleus.

Adrenal Gland Neoplasms↗
PubMed1999

Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila.

The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known (beta)-spectrin isoforms: a conventional (beta)-spectrin chain, and the novel isoform (beta)(Heavy)-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two (beta)-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing (beta)(Heavy)-spectrin into which the basolateral (beta)-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of (beta)-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of (beta)(Heavy)-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms.

Animals↗
PubMed1991

Oxidation of spectrin and deformability defects in diabetic erythrocytes.

We reasoned that de novo oxidative damage, as a result of increased protein glycosylation, could participate in the mechanisms whereby diabetic erythrocytes acquire membrane abnormalities. To examine this hypothesis, the extent of erythrocyte membrane protein glycosylation and the oxidative status of spectrin, the major component of the erythrocyte membrane skeleton, were examined. Labeling erythrocyte membranes with [3H]borohydride, which labels glucose residues bound to proteins, revealed that several proteins were heavily glycosylated compared with nondiabetic erythrocyte membranes. In particular, the proteins beta-spectrin, ankyrin, and protein 4.2 were the most glycosylated. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis of diabetic erythrocyte membranes did not reveal any quantitative or qualitative abnormalities in spectrin or other membrane proteins, examination of spectrin oxidative status by amino acid analysis and with cis-dichlorodiammineplatinum(II) (cDDP), a chemical probe specific for protein methionine and cysteine residues, demonstrated that the diabetic spectrin was oxidatively damaged: spectrin from diabetic subjects contained 35% less methionine (P less than 0.002), 15% less histidine (P less than 0.006), and a twofold increase in cysteic acid (P less than 0.001) compared with normal spectrin. Diabetic spectrin bound 32% less cDDP than normal spectrin (P less than 0.001); the lowest cDDP binding was observed with spectrin from insulin-dependent diabetic subjects. The extent of cDDP binding to diabetic spectrin correlated moderately and inversely with glycosylated hemoglobin (GHb) levels (n = 12, r = -0.727). Erythrocyte deformability, measured by ektacytometry, was decreased between 5 and 23% of control measurements (average of approximately 10%) in 21 of 32 diabetic subjects surveyed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1988

Spectrin and related molecules.

This review begins with a complete discussion of the erythrocyte spectrin membrane skeleton. Particular attention is given to our current knowledge of the structure of the RBC spectrin molecule, its synthesis, assembly, and turnover, and its interactions with spectrin-binding proteins (ankyrin, protein 4.1, and actin). We then give a historical account of the discovery of nonerythroid spectrin. Since the chicken intestinal form of spectrin (TW260/240) and the brain form of spectrin (fodrin) are the best characterized of the nonerythroid spectrins, we compare these molecules to RBC spectrin. Studies establishing the existence of two brain spectrin isoforms are discussed, including a description of the location of these spectrin isoforms at the light- and electron-microscope level of resolution; a comparison of their structure and interactions with spectrin-binding proteins (ankyrin, actin, synapsin I, amelin, and calmodulin); a description of their expression during brain development; and hypotheses concerning their potential roles in axonal transport and synaptic transmission.

Animals↗
PubMed2001

Studies of the erythrocyte spectrin tetramerization region.

Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T. A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.

Amino Acid Sequence↗
PubMed2005

Spectrin and ubiquitination: a review.

This review covers the observations leading to the conclusion that erythrocyte spectrin is a chimeric E2/E3 ubiquitin conjugating/ligating enzyme and the impact of this activity on the cell. Spectrin is important for the shape and the physical properties of the red blood cell, such as deformability and resistance to mechanical stress. The involvement of RBC spectrin in the ubiquitination process has been demonstrated. Human erythrocyte alpha-spectrin can facilitate formation of ubiquitin-spectrin adducts and conjugates in cell free systems (28). Computer analysis revealed domains that contained significant homologies to known consensus catalytic E2 and E3 sequences, and allowed us to develop a model for alpha-spectrin ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) enzymatic activities. The model has been tested and the precise E2/E3 site(s) identified by site-specific mutational analyses using a GST-fusion alpha-spectrin(2005-2415) recombinant in an in vitroubiquitination assay (26). The results indicated that cysteine 2071 and cysteine 2100 are critical for alpha-spectrin(2005-2415) E2/E3 activity as expected. However, both Cys2071 and Cys2100 are capable of transferring ubiquitin from an E1 enzyme to target sites within alpha-spectrin(2005-2415). This revealed a redundancy of function for human RBC spectrin's chimeric E2/E3 ubiquitin conjugating/ligating activity. Since spectrin is the major structural component of the erythrocyte membrane skeleton, and it constitutes 20% of the total RBC membrane protein, its ubiquitination enzymatic activity could play an important role in both erythropoietic cells and mature RBCs. This could also be one reason for evolving this redundancy of function.

Humans↗
PubMed1991

Spectrin isoforms in the mammalian retina.

Spectrin is a major component of the mammalian neuronal cytoskeleton. In the CNS, three isoforms of brain spectrin are known to exist: a cellular and dendritic isoform, (240/235E), related to neurons and glia; a cellular and axonal isoform, (240/235), related to neurons; and an isoform specific for astrocytes, (240/235A). In the present study, brain spectrins (240/235E) and (240/235) were localized within the mouse retina and optic nerve. Immunoblot analyses of proteins isolated from mouse retinas utilizing polyclonal antibodies to either brain spectrin (240/235) or brain spectrin (240/235E) revealed that these spectrins are present in the retina and that the two isoforms are the same molecular weights as those found in the brain. Immunocytochemical studies revealed that spectrin (240/235E) was localized in cell bodies of the inner nuclear, outer nuclear, and ganglion cell layers, and processes arborizing within the inner and outer plexiform layers. Spectrin (240/235) was distributed diffusely within the retina, lightly staining neurons in both the inner nuclear and outer nuclear layers, and the ganglion cell layer. In contrast to the situation found in the brain, spectrin (240/235) was but one of the axonal forms in the retina. We found that spectrin (240/235E) was also present in the axon-rich fiber layer and in the optic nerve and was often associated with fibrous elements. Spectrin (240/235) was also detected in the nerve fiber layer and optic nerve, but this isoform was not localized to fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Formation and properties of spectrin containing a truncated beta-chain, generated by an endogenous calcium-dependent protease.

Spectrin chromatographically isolated from human red cell membranes contains a proteolytic activity, inhibited by leupeptin, with a dependence on calcium ions characteristic of a calpain I. The activity accompanies the spectrin on two successive gel filtration columns and is present in both the dimer and the tetramer. It is cryptic in the ghost membrane and in purified membrane skeletons until these are dissociated, apparently because the spectrin is resistant to attack in the tetrameric state. The activity is more strongly expressed in spectrin preparations from young than from old cells. Since the bound enzyme does not detectably change the elution volume of spectrin in gel filtration, it is likely that its molecular weight is low. Its activity reveals itself only in the formation of a modified spectrin, lacking the COOH-terminal, phosphorylated portion of the beta-chain. The phosphorylated fragment (15-20 kDa) can be identified by gel electrophoresis. The proteolytically modified spectrin dimers are unable to associate with like molecules, but are univalent with respect to formation of hybrid tetramers with native spectrin. The association constant for this process is lower than that for self-association of native spectrin by a factor of only 2. The proteolytically modified spectrin behaves similarly in this respect to a truncated spectrin mutant found in a form of hereditary elliptocytosis. The latter also resembles the proteolytic product in that both give rise to the same altered NH2-terminal tryptic fragment from the alpha-chain.

Autoradiography↗
PubMed1986

Immunofluorescent patterns of spectrin in lymphocyte cell lines.

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.

Animals↗
PubMed1986

Spectrin subtypes in mammalian brain: an immunoelectron microscopic study.

Spectrin is a major cytoskeletal component of the brain. At least 2 distinct spectrin subtypes are found in mammalian brain: brain spectrin(240/235) and brain spectrin(240/235E). In the present study spectrin subtypes were localized in the adult mouse brain by immunoelectron microscopy using antibodies that recognize each subtype. Brain spectrin(240/235E) was concentrated in neuronal cell bodies, dendrites, and postsynaptic terminals. It was also prominently associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum, and nuclear envelope, and it appeared to interconnect structural elements within the cell. Brain spectrin(240/235E) also was localized to the plasma membrane, nuclear envelope, and cytoplasmic organelles of glial cell bodies. Brain spectrin(240/235) was detected in axons and presynaptic elements, where it was associated with the plasma membrane, microtubules, filaments, synaptic vesicles, and mitochondria. These results show that spectrin is distributed throughout the cytoplasm of neural cells, the location of spectrin is dependent on subtype, and the cytoplasmic surface of plasma membrane and organelles contains an extensive and intricate spectrin meshwork.

Animals↗
PubMed1984

Hemoglobin enhances the self-association of spectrin heterodimers in human erythrocytes.

Spectrin in isolated erythrocyte membranes is known to undergo tetramer to dimer transformation upon hypotonic incubation at 37 degrees C. In the present study, we detect no such transformation in intact erythrocytes in which hypotonicity is achieved by valinomycin treatment followed by hypotonic swelling. The inhibition of spectrin tetramer to dimer transformation is attributable to intracellular hemoglobin, since the addition of hemoglobin to isolated membranes or spectrin extracts blocks a similar spectrin transformation. However, the inhibitory effect is not limited to hemoglobin; other proteins including heme-containing proteins and basic proteins such as cytochrome c, ribonuclease, and albumin are also effective. The magnitude of their effect is proportional to the increased pI value of these proteins. We conclude that the stabilizing effect of these proteins on spectrin tetramers under hypotonic conditions is partly due to their non-ideality, which excludes water from spectrin and thus increases the effective concentration of spectrin, and to their electrostatic interactions with spectrin. In addition, promotion of spectrin self-association by hemoglobin under hypotonic conditions increases the stability of membrane skeletons against mechanical shearing. More importantly, the hemoglobin effect on spectrin self-association is demonstrable at physiological hemoglobin concentration, pH, and osmolarity, suggesting that in intact red cells the spectrin dimer-dimer association, as well as the membrane skeletal structure, is strengthened by intracellular hemoglobin.

Chromatography, Gel↗
PubMed1981

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.

Adenosine Triphosphatases↗