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At least 1,171 records · Page 65Linked to original sources
PubMed1983

Myotonic dystrophy: fragility of band 3 membrane protein upon resealing of erythrocyte ghosts and the normality of ATPase activity, sialic acid content, and spectrin extractability of ghosts.

Since a number of recent reports (2,14) have shown that myotonic dystrophy (MyD) might be an inherited disorder of cell membranes, we studied erythrocytes of 10 patients with MyD to investigate the membrane abnormality. In the osmotical aspect, the relative hemolysis of erythrocytes in patients with MyD in a solution of 75 mM NaCl displayed almost half the value of the normal level. Our experiments using electrophoresis demonstrated that a main polypeptide of membrane, band 3 (by the nomenclature of Fairbanks et al. (15], in patients with MyD was very fragile in the process of erythrocyte ghosts resealing in the isotonic medium. No significant differences were observed in membrane-bound Ca2+-ATPase activities, sialic acid contents, and spectrin extractability assessed by SDS-polyacrylamide gel electrophoresis between normal and MyD. It is suggested that abnormality of MyD erythrocytes may be localized to the surface membrane, especially around band 3.

Adult↗
PubMed1988

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

2-Amino-5-phosphonovalerate↗
PubMed1985

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro.

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.

Animals↗
PubMed1982

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin. A spectrin-like calmodulin-binding protein (fodrin).

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated Mr of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca2+ and calmodulin. Peak II revealed a basal activity in the absence of Ca2+ and calmodulin, which was stimulated 2-fold by addition of Ca2+. Calmodulin had no effect on the peak II activity.

Animals↗
PubMed1986

Inhibition of protein synthesis by the beta-subunit of spectrin.

The 220 kDa beta-subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme-regulated eIF-2 alpha kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF-2 on its alpha-subunit.

Animals↗
PubMed1987

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.

Actins↗
PubMed1989

Interferons as gene activators: a cluster of six interferon-activatable genes is linked to the erythroid alpha-spectrin locus on murine chromosome 1.

Several interferon-activatable murine genes were mapped to murine chromosomes by hybridizing cDNA probes to Southern blots of genomic DNA samples from a panel of mouse-hamster somatic cell hybrid lines. The 12 gene is located on chromosome 12 and it specifies a 3.6-kb mRNA. The 204 gene (specifying a 2.5-kb mRNA), and three genes of the 203 gene family (hybridizing to five mRNAs of sizes between 2 and 4.5 kb), together with the 202 gene (specifying a 2-kb mRNA) are located on murine chromosome 1. By restriction fragment length polymorphism analysis of DNA samples prepared from a panel of recombinant inbred mouse lines (C57BL/6J D DBA/2J) and from 85 [C3H/HeJ-gld/gld x Mus spretus) F1 X C3H/HeJ-gld/gld] backcross mice we established a close linkage of the 202, 203, and 204 genes to the erythroid alpha-spectrin gene (Spna-1) on distal murine chromosome 1. Cosmids containing the 202, 203, and 204 genes were isolated from a library derived from AKR mouse DNA. Southern blot analysis of such cosmids revealed: (a) hybridization of a partial 203 cDNA to three genes of the 203 gene family; (b) cross-hybridization of the 202 and 204 genes with one another and with a third gene (designated as 201 gene), and (c) a close linkage of genes of the 203 family with the 201, 202, and 204 genes. These results indicate the existence of a cluster of at least six closely linked, interferon-activatable genes on distal murine chromosome 1 in the vicinity of the Spna-1 locus and also of the Minor lymphocyte stimulating locus (Mlsa).

Animals↗
PubMed1983

Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border.

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (approximately 5 nm wide, 100-200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deep-etch technique, these approximately 5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of approximately 5 nm fibrils that are morphologically distinct from the approximately 8 nm myosin-containing fibrils which were identified by anti-myosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actin-vesicle, actin-intermediate filament and vesicle-plasma membrane linkages.

Animals↗
PubMed1995

The ultrafiltration and ESR study of Cd2+ binding to human erythrocyte spectrin.

The Cd2+ binding to human erythrocyte spectrin tetramer (SPT) was studied by the ultrafiltration method. The results indicate that the Cd2+ binding depends on Cd2+ concentration in a biphasic feature. Thus, it is different from the Tb(3+)-SPT reaction. The biphasic feature is in accordance with the results of fluorescence and circular dichroism (CD) studies on Cd(2+)-SPT complex. The data were analyzed by the Scatchard method. At the first stage (the mole ratio of Cd2+/SPD < 45), there are two types of Cd2+ binding sites, with the binding sites and corresponding binding constants determined as: n1 = 6, K1 = 3.3 x 10(5) M-1; n2 = 8, K2 = 9.1 x 10(4) M-1. At the second state (Cd2+/SPD > 45), the Cd2+ binding manifests a positive cooperative effect. The interaction between Cd2+ and maleimide spin labeled (MSL) SPT was studied by the ESR method. The results were fitted with the multiple equilibrium model, and one Cd2+ high-affinitive binding site with association constant 9.61(7) x 10(5) M-1 was obtained. The conjugation of maleimide with thiol group results in the decrease of Cd2+ high-affinitive binding sites from 6 to 1. This demonstrated that the thiol groups of SPT were involved in the high binding sites. The increase of mobile fraction of MSL in low Cd2+ concentration revealed that Cd2+ binding to SPT induces a significant conformation change of MSL-SPT. The rotation correlation time of MSL attached to SPT varied from 4.4 x 10(-9) to 6.5 x 10(-9) sec.

Binding Sites↗
PubMed1991

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

Actins↗
PubMed1986

Studies on the structural polymorphism of the alpha-II domain of human erythrocyte spectrin.

Following restricted tryptic digestion at 4 degrees C, a structural polymorphism affecting the alpha-chain of human spectrin, the major erythrocyte membrane skeleton protein, has recently been described in American blacks (Knowles, W.J., Bologna, M.L., Chasis, J.A., Marchesi, S.L. and Marchesi, V.T. (1984) J. Clin. Invest 73, 973-979). Four variants affecting the alpha-II domain or its tryptic products have been characterized, depending on changes in molecular weight and/or isoelectric point. One variant of the alpha-II domain (Type 2) shows an increase in apparent molecular weight and basic shift in pI. It contains a limit chymotryptic peptide showing a change in chromatographic mobility on two-dimensional electrophoresis which is thought to reflect a sequence alteration associated with the increase in apparent molecular weight. We find that this altered limit chymotryptic peptide is not unique to the Type 2 variant, but is also present in a variant (Type 4) showing only the same basic shift in pI as the Type 2 variant. It is not found in a variant (Type 3) showing only an increase in apparent molecular weight. The most likely explanation for these findings is that the altered limit chymotryptic peptide common to both the Type 2 and Type 4 variants is responsible for the change in isoelectric point which is common to both these variants. An as yet unidentified change elsewhere in the polypeptide chain must be responsible for the observed alteration in molecular weight of the Types 2 and 3 variants.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.

Erythrocyte Membrane↗
PubMed1995

Proteolysis of spectrin by calpain accompanies theta-burst stimulation in cultured hippocampal slices.

Tests were carried out to determine if repetitive bursts of afferent stimulation activate calpain, a calcium-dependent protease hypothesized to be involved in the production of long-term potentiation. Antibodies against a stable breakdown product that results from proteolysis of spectrin by calpain were used to identify sites of enzyme activation in cultured hippocampal slices. Slices in which theta-burst stimulation was applied to the Schaffer collateral fibers had pronounced accumulations of breakdown product that were restricted to field CA1, the zone innervated by the stimulated axons. Labelling occurred in the form of scattered puncta and was also present in dendritic processes. The extent of these effects was correlated (r = 0.73) with the amount of theta-burst stimulation delivered. Control slices or those receiving low frequency stimulation had variable, but uniformly lower, amounts of breakdown product and were clearly distinguishable from those given theta bursts. Statistical analyses using a six point rating scheme confirmed this point (P < 0.001). These results satisfy an essential prediction of the hypothesis that calpain plays an important role in the induction of long-term potentiation.

Afferent Pathways↗
PubMed1982

Solution scattering studies of dimeric and tetrameric spectrin.

The structure of spectrin dimers and tetramers in solution has been examined by light, low-angle X-ray and neutron scattering. The results show a good correspondence between the solution dimensions of these molecules and their appearance in the electron microscope after shadowing. The scattering profiles are not compatible with an extended rod-like character, but reflect the presence of a considerable degree of bending. The radii of gyration of the dimer and tetramer were determined to be 170 and 375 A and the cross-section radii of gyration 14 and 12.3 A, respectively. Both are thus long, thin, rather bent molecules, and the tetramer is twice the length of the dimer.

Humans↗
PubMed1994

Reinvestigation of the thermodynamics of spectrin self-association.

The thermodynamics of the self-association reactions of human spectrin have been reinvestigated by means of sedimentation equilibrium over the temperature range 18-40 degrees C. The experimental data were analysed in terms of a cooperative isodesmic model of association. The van't Hoff plot showed that the standard change in enthalpy for the heterodimer-tetramer step was temperature-dependent, leading to an estimate of -8.5 kJ mol-1 K-1 for the change in molar heat capacity, delta Cp. Curvature in the van't Hoff plots, not detected in previous studies, was revealed through the increased precision of the data and the wider temperature range examined. On the assumption that delta Cp reflects hydrophobic interactions in the tetramer that cannot be formed in the heterodimer, it can be estimated that approximately 50 CH2 groups per heterodimer participate in hydrophobic interactions in the tetramer that cannot be formed in the heterodimer.

Calorimetry↗
PubMed1986

Insulin inhibits the phosphorylation of the membrane cytoskeletal protein spectrin in pig erythrocytes.

Incubation of either ghost membranes with 32P- -ATP or intact erythrocytes with 32P-inorganic phosphate led to phosphorylation of the beta-chain of the major membrane-associated protein spectrin. This phosphorylation was reduced by 30% by insulin (10-100 microU/ml) both in membranes and in intact cells. The results show that the membrane-cytoskeleton is responsive to extracellular signals such as hormone receptor activation.

Animals↗
PubMed1986

A rapid purification of synapsin I: a neuron specific spectrin binding protein.

We have developed a one chromatographic step isolation protocol for the neuron specific protein synapsin I. This procedure results in a yield of 80 micrograms/g brain, which is ten fold better than the highest yield yet reported for this protein. The authenticity of the synapsin I isolated by this procedure is demonstrated by comigration with authentic synapsin I on SDS-polyacrylamide gels, crossreactivity with antibody specific against synapsin I, and nearly identical two dimensional chrymotryptic iodopeptide maps of authentic synapsin I and the protein purified by this protocol. Synapsin I isolated by this procedure retains its functional properties, demonstrated by the ability of synapsin I to stimulate the formation of a brain spectrin(240/235)/synapsin I/F-actin ternary complex as determined by a low shear falling ball viscometry assay. This novel protocol therefore has the advantage of being a rapid, high yield procedure that retains the functional properties of synapsin I.

Actins↗