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At least 865 records · Page 48Linked to original sources
PubMed1995

Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas cells.

It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardtii contain polypeptides cross-reacting with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either alpha- or beta-spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent.

Animals↗
PubMed2000

A putative spectrin-containing membrane skeleton in hyphal tips of Neurospora crassa.

The apical plasma membrane (PM) is important in hyphal tip growth, where it may regulate tip extensibility via its association with an appropriate membrane skeleton (MS). By cell fractionation and immunocytochemistry we show that proteins with characteristics of actin, spectrin, and integrin are associated in a MS-like manner with the PM of Neurospora crassa hyphae. The spectrin-like protein in particular is highly concentrated at the PM in the region of maximum apical expansion. This protein shares with other spectrins immunoreactivity, molecular weight, PM association, and actin binding capacity. Its distribution in hyphae suggests that it is a dominant component of the MS in true fungi and is critical to hyphal tip growth.

Cell Fractionation↗
PubMed1999

Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.

Animals↗
PubMed1993

Spectrin alpha IIa variant in dominant and non-dominant spherocytosis.

Several polymorphic mutations are located on the spectrin alpha-chain; among these the variant termed alpha IIa is characterized by an acid shift in the isoelectric point of the tryptic digest peptides 46 kDa and 35 kDa. In this variant a single amino acid substitution (alanine to aspartic acid) occurred at position 972 of the spectrin alpha-chain due to a point mutation (GCT to GAT) in the DNA. This variant, which seemed very rare in normal people, could be related to the recessive form of hereditary spherocytosis (HS) and could be absent in the dominant form of the disease. We have studied the alpha IIa variant by denaturing electrophoresis of the spectrin tryptic digest peptides from 179 subjects: 46 controls, 78 patients with dominant (d) or non-dominant (nd) HS and 55 relatives of the patients. The confirmation of the results was obtained at the DNA level in 41 subjects. The frequency of the chromosome bearing the alpha IIa mutation was 7.6% in controls and higher (about 12-14%) in members of families with dHS as well ndHS. However, the family trees clearly showed that the mutation and the HS disease gene(s) were located on different chromosomes and inherited independently from each other. Furthermore, our study allows the conclusion that in most (if not all) cases of dHS, the alpha IIa the variant is not the cause, is not a marker, and does not influence the phenotypic expression of the disease.

Adult↗
PubMed1992

Effect of Cu(2+)-ascorbic acid on lipid peroxidation, Mg(2+)-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin.

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu2+ and ascorbate in vitro, Mg2+ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu2+ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg(2+)-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg(2+)-ATPase activity and partially recovers spectrin of RBC membrane.

Animals↗
PubMed1988

The Duffy blood group is linked to the alpha-spectrin locus in a large pedigree with autosomal dominant inheritance of Charcot-Marie-Tooth disease type 1.

The alpha-spectrin locus (SPTA) on chromosome 1 maps to 1q22-q25 and alpha-spectrin specific probes detect restriction fragment length polymorphisms (RFLPs) with the endonucleases MspI and PvuII. The Duffy blood group (FY) has been mapped to the 1p21-q23 region. We found positive linkage between the alpha-spectrin and the Duffy loci with a maximal Lod score of 3.81 at theta = 0.0 using the computer program MLINK. This indicates that both loci are very closely linked and probably localized to 1q22-q23.

Blood Group Antigens↗
PubMed1989

Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of myelinated nerve fibres.

The myelin-associated glycoprotein (MAG) is an intrinsic membrane protein that is specific for myelinating cells. MAG has been proposed to function in the PNS as an adhesion molecule involved in Schwann cell-axon contact and maintenance of cytoplasmic channels within the myelin sheath. In this report we show that the microfilament components, F-actin and spectrin, co-localize with MAG in periaxonal membranes, Schmidt-Lanterman incisures, paranodal myelin loops, and inner and outer mesaxons of myelinating Schwann cells. F-actin was localized light microscopically by rhodamine-labelled phallicidin binding. Spectrin and MAG were localized by light microscopic and ultrastructural immunocytochemistry. The findings indicate that plasma membrane linkage of F-actin in Schwann cells is likely to occur via spectrin, and raise the possibility that microfilaments interact with the cytoplasmic domain of MAG. An interaction between MAG and microfilaments would be consistent with the proposed function of MAG as an adhesion molecule.

Actins↗
PubMed2005

Spectrin and calpain: a 'target' and a 'sniper' in the pathology of neuronal cells.

It is well documented that activation of calpain, a calcium-sensitive cysteine protease, marks the pathology of naturally and experimentally occurring neurodegenerative conditions. Calpain-mediated proteolysis of major membrane-skeletal protein, alphaII-spectrin, results in the appearance of two unique and highly stable breakdown products, which is an early event in neural cell pathology. This review focuses on spectrin degradation by calpain within neurons induced by diverse conditions, emphasizing a current picture of multi-pattern neuronal death and a recent success in the development of spectrin-based biomarkers. The issue is presented in the context of the major structural and functional properties of the two proteins.

Aging↗
PubMed1999

Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin.

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the alpha-chain and the C-terminus of the beta-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the beta-chain and one helix from the N-terminus of the alpha-chain. Three mutant beta-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The alpha- and beta-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded beta-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phenomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wildtype alpha- and beta-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in alpha-helicity. From continuous-variation profiles an association constant in the range 1-2 x 10(6) M-1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin beta-chain. Of the three mutations in the beta-chain fragment, one (an Ala-->Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the alpha-chain fragment, whereas Trp-->Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations.

Binding Sites↗
PubMed2006

Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa.

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

Hyphae↗
PubMed1987

Comparison of thermal properties of bovine spectrin and fodrin.

Thermal properties of bovine brain fodrin have been studied by circular dichroism and electron spin resonance and compared to those of bovine erythrocyte spectrin. Protein unfolding was induced either by urea or by a combination of heat and urea. The denaturation profiles of the two proteins, as measured by the changes in ellipticity at 222 nm as a function of temperature, were very similar but fodrin denaturation occurred at both higher temperatures and higher urea concentrations. In the absence of urea the thermal transition of spectrin was centered at 51 degrees C and that of fodrin at 54.5 degrees C. Proteins were also labeled with a maleimide analog spin probe. Spin-labeled fodrin showed a thermal transition similar to that of spectrin but centered at 46 degrees C instead of 42 degrees C. These findings indicated a close structural similarity of the two proteins but a slightly higher conformational stability of fodrin to both heat and urea.

Animals↗
PubMed1978

Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release.

Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15 degrees C of higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 micron for Ca2+ and 200 micron for Mg2+. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca2+, loss of spectrin does not occur. These results suggest that an intact spectrin netwoek is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.

Calcium↗
PubMed1978

Selective alteration of erythrocyte deformabiliby by SH-reagents: evidence for an involvement of spectrin in membrane shear elasticity.

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reaents on the deformability of human erythrocytes was studied. Deformability was quuantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N, N' -p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Anaalysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides. The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.

Diamide↗
PubMed1985

Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe.

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Human erythrocyte spectrin dimer intrinsic viscosity: temperature dependence and implications for the molecular basis of the erythrocyte membrane free energy.

We have determined experimentally the temperature dependence of human erythrocyte spectrin dimer intrinsic viscosity at shear rates 8-12 s-1 using a Cartesian diver viscometer. We find that the intrinsic viscosity decreases from 43 +/- 3 ml/g at 4 degrees C to 34 +/- 3 ml/g when the temperature is increased to 38 degrees C. Our results show that spectrin dimers are flexible worm-like macromolecules with persistence length about 20 nm and that the mean square end-to-end distance for this worm-like macromolecules decreases when the temperature is increased. This implies that the spectrin dimer internal energy decreases when the end-to-end distance is increased and that the free energy increase associated with making the end-to-end distance longer than the equilibrium value for the free molecules is of entropic origin. The temperature dependence of the erythrocyte membrane shear modulus reported previously in the literature therefore appears mainly to be due to temperature dependent alterations in the membrane skeleton topology.

Erythrocyte Membrane↗
PubMed1987

Heat-induced dissociation of human erythrocyte spectrin dimer into monomers.

Human erythrocyte spectrin heated above 49 degrees C could be separated into two fractions by DEAE-Toyopearl column chromatography at room temperature. The first fraction eluting with the salt gradient was predominantly the alpha subunit, indicating a heat-induced dissociation of the spectrin alpha beta dimer into monomers. The second fraction, obtained with 0.5 M NaOH after salt elution, consisted of high-molecular-weight proteins in addition to alpha and beta subunits, which were visualized by gel electrophoresis with sodium dodecyl sulfate. The isolated beta subunit when heated above 48 degrees C could also be separated into two fractions by column chromatography. About 30% of the protein eluted with the salt solution and the rest of the proteins were in the alkali eluate in which high molecular weight protein bands also appeared, indicating a heat-induced aggregation of the beta subunits. Almost all the isolated alpha subunit, however, eluted out with the salt solution, even though the subunit was heated at 52 degrees C. Studies of the binding of subunits to inside-out vesicles indicate that the isolated beta subunit was denatured irreversibly by heating; on the other hand, the alpha subunit kept its binding ability after heating above 50 degrees C. These findings are attributed to the heat-induced dissociation of the spectrin molecules into alpha and beta subunits at 49-50 degrees C, and eventual aggregation of the denatured beta subunits.

Chromatography, Gel↗
PubMed1988

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin.

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1979

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.

Circular Dichroism↗