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PubMed2005

Haemolysis caused by alterations of alpha- and beta-spectrin after 10 to 35 min of severe exercise.

The pathophysiology of exercise related haemolysis is not thoroughly understood. We investigated whether exercise related haemolysis (1) is associated with alterations of red blood cell (RBC) membrane proteins similar to those found in inherited anaemic diseases, (2) can be induced with a non-running exercise mode, (3) is related to exercise intensity, and (4) coincides with indicators of oxidative stress. In ten triathletes [median (P25/P75-percentiles) age: 28.0 (26.3/28.5) years, height: 1.84 (1.78/1.87) m, body mass: 78.5 (74.8/80.8) kg, maximal oxygen uptake: 60.0 (57.3/64.8) ml kg(-1) min(-1)], haptoglobin, alpha- and beta-spectrin bands, malondialdehyde (MDA) and H2O2-induced chemiluminescence (H2O2-Chem) were determined immediately pre- and post-both, a 35 min low intensity and a high intensity cycling exercise [240 (218/253) vs 290 (270/300) W, P<0.05) requiring similar amounts of metabolic energy [28.3 (25.9/29.9) vs 24.9 (18.4/30.5) kJ kg(-1), P>0.05]. At high exercise intensity haptoglobin [1.10 (0.81/2.53) vs 1.01 (0.75/2.00) g l(-1)] decreased (P<0.05) whilst MDA [2.80 (2.65/3.20) vs 3.13 (2.78/3.31) nmol ml(-1)] and H2O2-Chem [29.70 (22.55/37.10) vs 37.25 (35.20/52.63) rel. U min] increased (P<0.05), coinciding with the disappearance of the spectrin bands in six out of ten gels. No corresponding changes were found at low intensity exercise. Ten to 35 min of non-running exercise in a regularly used intensity domain causes intra-vascular haemolysis associated with alterations in the RBC membrane proteins similar to those found after in vitro oxidative stress and in inherited anaemic diseases like Sphaerocytosis and Fanconi's anaemia.

Adult↗
PubMed1976

Physico-chemical characterization of the spectrin tetramer from bovine erythrocyte membranes.

The tetramer of bovine spectrin has been purified and characterized in terms of its hydrodynamic and optical properties. (1) The molecular weight, from both sedimentation equilibrium and sedimentation velocity/diffusion measurements, is close to one million. (2) The hydrodynamic properties suggest a highly expanded but basically symmetrical molecule of Stokes radius 200 A. (3) Optical rotatory dispersion measurements indicate a high degree of order in the tertiary structure of the molecule. These results are not consistent with the assumption that is often made, that the spectrin molecule is a long fibrous rod.

Animals↗
PubMed1981

The molecular basis of the defect in phosphorylation of spectrin in human hereditary spherocytosis.

The molecular basis for the depressed phosphorylation of the smaller polypeptide of spectrin (band 2) in the erythrocytes of patients suffering from hereditary spherocytosis is investigated. Comparison of healthy and spherocytic spectrin polypeptides by controlled proteolysis reveals no abnormality in the degradation pattern or in the sites of phosphorylation. It is concluded that the lesion is a consequence of a defective control of phosphorylation. The defect can be mimicked in healthy cells by the introduction of calcium into the erythrocyte and the possibility that the primary pathological lesion is a deficient control of the calcium content of the erythrocyte is discussed.

Adult↗
PubMed1981

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes.

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.

Casein Kinases↗
PubMed1984

Phospholipid asymmetry in the membranes of intact human erythrocytes and in spectrin-free microvesicles derived from them.

Phospholipase A2 from bee venom and Naja naja has been used to study the orientation of phospholipids present in the membrane of intact human erythrocytes and in spectrin-free microvesicles derived from the cells by treatment with Ca2+ and A23187. Little difference between the cells and microvesicles was observed in the apparent accessibility of phospholipids to the enzyme, suggesting that the original lipid asymmetry was maintained in the absence of spectrin. However, incubation of the microvesicles for 16 h at 37 degrees C did lead to partial loss of asymmetry in the transmembrane distribution of phosphatidylcholine and phosphatidylethanolamine but not of phosphatidylserine. Despite the similarity of lipid asymmetry in cells and fresh microvesicles, the latter were about 40-fold more sensitive to phospholipase treatment than were cells. Although they retained the lipid asymmetry of intact cells, the microvesicles resembled ghosts in their great sensitivity to phospholipase A2 attack, suggesting that the lipid packing in microvesicles and ghosts was similar. This conclusion was supported by the results of experiments with a fluorescent probe Merocyanine 540.

Animals↗
PubMed1989

Association of hemin with protein 4.1 as compared to spectrin and actin.

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.

Actins↗
PubMed1991

EPR study of the hydrophobic interaction of spectrin with fatty acids.

The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in binding studies.

Binding Sites↗
PubMed1992

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

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

Cell Membrane Permeability↗
PubMed1995

Neutral polymers elicit, and antibodies to spectrin, band 4.1 protein and cytoplasmic domain of band 3 protein inhibit the concanavalin A-mediated agglutination of human erythrocytes.

Concanavalin A (Con A) is known to agglutinate human erythrocytes if the cells are pre-treated with a proteinase or neuraminidase. We report that untreated cells can also be made to agglutinate with the lectin if the lectin-bound cells are treated with anti-Con A antibodies, or if a neutral polymer such as serum albumin, polyvinylpyrrolidone or Ficoll is added. Thus, Con A falls in the category of 'incomplete' lectins. The polymer induces Con A-agglutinability without altering the receptor number, or deformability of the cells. If the polymer is sequestered within erythrocyte ghosts, Con A is unable to agglutinate them; but the presence of the polymer only on the outer surface (as in intact cells) or on both the surfaces permits agglutinability. Thus, the site of the polymer effect resides on the outer surface of the membrane. The polymer, however, is unable to induce agglutinability in erythrocyte vesicles, whose membrane lacks skeletal proteins. The result suggests a positive role for the membrane skeleton in the process of agglutination brought about by the polymer, as is true also for the agglutination of proteinase-treated cells. In order to obtain detailed information on the proteins participating in agglutination, monospecific antibodies to spectrins, band 4.1 protein, ankyrin and the cytoplasmic domain of band 3 protein were internalized in erythrocytes. It is found that anti-spectrin and anti-band 3 cytoplasmic domain, but not their Fab's, inhibit the Con A-mediated agglutinability partially, and anti-4.1 antibodies, as well as the Fab's, inhibit the agglutinability substantially. Anti-ankyrin, however, was without any effect. The results confirm a positive role for the membrane skeleton in the Con A-mediated agglutination of normal erythrocytes in the presence of a neutral polymer, or in proteinase treated cells. We also provide evidence for requirement of Mg-ATP in the agglutination process.

Adenosine Triphosphate↗
PubMed1996

Phospholipid asymmetry in red blood cells and spectrin-free vesicles during prolonged storage.

Erythrocytes and spectrin-free DMPC-induced vesicles released from the cells were incubated for 3 weeks at 6 degrees C under conditions of metabolic ATP-depletion. Phosphatidylserine (PS) asymmetry was monitored during this period by use of the prothrombinase assay. Prothrombinase activities measured at the beginning of the incubation period indicated that approximately 0.06% of PS was located at the outer layer of the red cell membrane, whereas in DMPC-induced vesicles approximately 1.5% the PS was exposed on the outside. After completion of the incubation period PS exposure on the outside of red cells and vesicles was increased by no more than 5-fold. On the other hand, with vesicles prepared with a significantly increased (4-fold) ATP-content to sustain translocase activity, the incubation process resulted in a surprisingly high (20-fold) increase of PS exposure. With vanadate, an inhibitor of the aminophospholipid translocase, included in the incubation medium, the redistribution of PS was even more pronounced. These observations indicate that PS asymmetry in spectrin-free vesicles can not be directly correlated to either ATP content or translocase activity and suggest that besides the aminophospholipid translocase and the membrane skeleton, other mechanisms must be involved in maintaining phospholipid asymmetry.

Adenosine Triphosphate↗
PubMed1978

Quantitative analysis of the amino-terminal residues of spectrin by use of the transamination reaction.

The metal ion-catalysed transamination reaction has been examined as a means of quantitative amino-terminal analysis of proteins. Application of this method to the erythrocyte membrane protein, spectrin, showed that this protein contained a single amino-terminal residue per 240,000 daltons. This value supports the hypothesis that spectrin is comprised of two polypeptide chains of approx. 220,000 and 250,000 daltons, respectively.

Amination↗
PubMed1983

The effect of cross-linking spectrin-actin complexes with band 4.1 on the state of polymerization of the actin.

The polymerization of actin in the presence of spectrin tetramers and band 4.1 isolated from the human erythrocyte has been measured using a fluorescence energy transfer technique. The results show that the cross-linking of spectrin-actin complexes by band 4.1 results in a limited depolymerization of actin filaments and a concomitant rise in the critical actin concentration. The phenomenon may explain in part the existence of actin in the erythrocyte cytoskeleton as short oligomers rather than as long filaments.

Actins↗
PubMed1984

On the mechanism for inactivation of cytochalasin binding activity associated with F-actin and spectrin-band 4.1-actin complex by sulfhydryl reagents.

The sulfhydryl group modifying reagent, p-hydroxymercuribenzoate, inhibited the cytochalasin binding activity of the actin nuclei in the spectrin-band 4.1-actin complex from the erythrocyte membrane and of muscle F-actin. Kinetic studies indicated that while the cytochalasin binding activity was immediately inhibited, the actin remained filamentous and depolymerized slowly over a period of 1 to 2 h. Scatchard analysis of the binding data revealed that initially only the KD was affected. However, prolonged incubation led to depolymerization of the F-actin and dissociation of the spectrin-band 4.1-actin complex, resulting in loss of binding sites. It thus appears that certain actin sulfhydryl group(s) are important for cytochalasin binding. However, the most reactive sulfhydryl group (cys-374) on actin does not appear to be involved.

Actins↗
PubMed1984

Interaction of anilinonaphtyl labeled spectrin with fatty acids and phospholipids: a fluorescence study.

Anilinonaphtyl labeled spectrin exhibits a fluorescence emission spectrum characteristic of a highly hydrophobic environment. Quenching of the fluorescence intensity by nitroxide analogs of fatty acids of affinity 10(4) M-1 reveals that the sites of interaction of fatty acids lie very close to the anilinonaphtyl groups. Similar experiments performed with a nitroxide analog of phosphatidylserine yield a 30% quenching of fluorescence while the same phosphatidylcholine analog has essentially no effect. The changes in the fluorescence emission spectrum exhibited in the presence of sonicated phosphatidylserine vesicles further outline the specificity of interaction towards phosphatidylserine, with one spectrin binding site per about 750 exposed phospholipids. Moreover, they suggest a penetration of the anilinonaphtyl group into the lipid bilayer.

Fatty Acids↗
PubMed1989

Analysis of the spectrin binding domains of globin.

This study describes the results of binding studies between human spectrin and peptides obtained by trypsin digestion of human globin. The globin digest when passed through an affinity column of spectrin-coupled sepharose retained one peptide from both alpha and beta chains of globin. The absorbed peptides were eluted with 4 M guanidine hydrochloride and separated on a reverse phase column by high pressure liquid chromatography. Their amino acid sequence was determined and their position located in the globin molecule.

Amino Acid Sequence↗
PubMed1981

Erythrocyte spectrin in Duchenne muscular dystrophy.

The primary structure of erythrocyte spectrin bands I and II from controls and patients with Duchenne muscular dystrophy was compared by 2-dimensional peptide mapping. 125I-labelling was done either by the chloramine-T method or using the Bolton and Hunter reagent followed by treatment with trypsin or chymotrypsin, resulting in four different peptide maps from each band of spectrin. Although all the peptide maps of band I were considerably different to those of band II, there were no consistent differences in the maps of bands I and II from controls compared to the corresponding maps from patients with Duchenne muscular dystrophy.

Chymotrypsin↗
PubMed1984

Spectrin and protein 4.1 as an actin filament capping complex.

Spectrin and protein 4.1, when added to G- or F-actin, cause the formation of short filaments, as judged by the appearance of powerful nucleating activity for G-actin polymerisation. F-Actin filaments are rapidly fragmented under physiological solvent conditions. The effect of cytochalasin E on the polymerisation reaction and the extent of reduction in the critical monomer concentration of actin when spectrin and 4.1 are added suggest that these proteins form a capping system for the more slowly growing, or 'pointed' ends of actin filaments. The interaction is not affected by calcium or by 4.9, the remaining constituent of the purified red cell membrane cytoskeleton.

Actins↗
PubMed1989

Structural basis for the high activation energy of spectrin self-association.

The association of spectrin hetero-dimer (alpha beta) to the tetramer (alpha 2 beta 2, which predominates in the cell) is marked by an exceptionally high activation energy, so that the reaction does not proceed measurably in the cold. We have tested the hypothesis that this is due to intra-dimer association between the alpha- and beta-chain ends, which must be broken before tetramers can form. Two mutant univalent spectrins with association defects at the alpha and beta ends, respectively, and incapable therefore of intra-dimer bonding, were found to associate rapidly with one another at 4 degrees C. The bimolecular rate constant is greater than for the association of normal dimers by 6 orders of magnitude.

Elliptocytosis, Hereditary↗