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PubMed1989

Frequency dependence of the shear moduli of spectrin studied using a multiple lumped resonator viscoelastometer.

The frequency dependence (119-7860 Hz) of the storage and loss shear moduli, G' and G'', of human erythrocyte spectrin dimer crude solutions at 22.5 degrees C has been measured using a Birnboim-Schrag multiple lumped resonator viscoelastometer. The measurements were carried out on solutions of ionic strength 1 mM containing 1.1-3.7 mg ml-1 spectrin. This corresponds to the terminal zone for G' and G''. Analysis of the data using the standard theory of hybrid relaxation spectra yields a relaxation time of 22.5 +/- 1 microseconds. The pure spectrin dimer relaxation time is estimated to be 16 +/- 3 microseconds. This result suggests that at an ionic strength of 1 mM, the spectrin dimers are extended and that the main relaxation process is simple end-over-end rotation.

Biophysics↗
PubMed1992

Characterization of calcium binding to brain spectrin.

Brain spectrin alpha and beta chains bind 45Ca2+, as shown by the calcium overlay method. Flow dialysis measurements revealed eight high affinity binding sites/tetramer that comprise two binding components (determined by nonlinear regression analysis). The first component has one or two sites (kd = 2-30 x 10(-8) M), depending on the ionic strength of the binding buffer, with the remaining high affinity sites in the second component (kd = 1-3 x 10(-6) M). In addition, there is a variable, low affinity binding component (n = 100-400, kd = 1-2 x 10(-4) M). Magnesium inhibits calcium binding to the low affinity sites with a K1 = 1.21 mM. Proteolytic fragments from trypsin or chymotrypsin digests of brain spectrin bind 45Ca2+ if they include alpha domain IV, alpha domain III, or the amino-terminal half of the beta chain (but more than 25 kDa from the amino-terminal). These data suggest that calcium ions bind with high affinity to the putative EF-hands in alpha domain IV and to one site in the amino-terminal half of the beta chain that is associated with alpha domain IV in the native dimer. The localization is consistent with a direct calcium modulation of the spectrin-actin-protein 4.1 interaction. In addition, there appears to be one high affinity site near the hypersensitive region of alpha brain spectrin. All four proposed binding sites occur near probable calmodulin-binding or calcium-dependent protease cleavage sites.

Animals↗
PubMed1990

Investigation of spectrin binding to phospholipid vesicles using isoindole fluorescent probe. Thermal properties of the bound and unbound protein.

Fluorescence of isoindole probe covalently bound to spectrin from pig erythrocytes, and fluorescence of tryptophanyl residues were used to study spectrin interaction with phospholipid bilayers. Evidence would be provided for conformational changes of spectrin occurring upon its binding to lipid bilayers. Fluorescence quenching experiments allowed to determine thermal stability of the protein in bound and unbound state. Spectrin binding to lipids was shown to protect the protein against thermal denaturation.

Animals↗
PubMed1989

Characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies.

An important function of the mammalian nonerythroid alpha-spectrin chain (alpha-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid alpha chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid alpha chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain alpha-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into alpha-helical conformations, these binding sites are predicted to form amphipathic structures.

Amino Acid Sequence↗
PubMed1989

[Family of hereditary elliptocytosis with abnormalities of spectrin function (Sp alpha 1/74)].

We have experienced a case of cytohemolytic hereditary elliptocytosis (HE) in a six-year-old boy. Metabolisms of the erythrocytic membrane were investigated on the members from his pedigree. The results were as follows; 1) The presence or absence of ovalocytic HE were studied in his pedigree. 2) Failure in the process of spectrin dimer to tetramer conversion was found. 3) Although abnormality existed in conversion of D to T by the patient's alpha-chain spectrin and normal beta-chain spectrin, no abnormality was recognized when normal alpha-chain and the patient's beta-chain were combined. 4) Decrease of the alpha-chain (80 kd) domain and appearance of abnormal (74 kd) spot were found by two dimensional peptide mapping of spectrin. 5) In his pedigree, neither patients with hereditary pyropoikilocytosis (HPP) nor carrier states were recognized. In summary, this patient's pedigree was considered to be HE [SP alpha 1/74]. This case appears to be the first case in Japan and only few cases have been reported in the world literature.

Child↗
PubMed1988

Interaction of spectrin with hydrophobic agaroses.

Purified spectrin was found to interact strongly with hydrophobic agaroses such as Phenyl- or Octyl-Sepharose, in the presence of EDTA. From the complexes formed spectrin was eluted with ethylene glycol but not with low ionic strength solutions. The binding capacity of spectrin increased with increasing ionic strength of the equilibration buffer and showed but little dependence on its pH value. The fragments obtained by proteolysis of spectrin carried out under mild conditions were also found to bind strongly to phenyl-agarose, and were eluted with ethylene glycol. The fractions eluted with ethylene glycol contained two closely related polypeptides of Mr 65,000 and 60,000.

Buffers↗
PubMed1988

Molecular cloning of the cDNA for human erythrocyte beta-spectrin.

Overlapping cDNA clones, totaling 3.3 kilobases (kb) in length, which encode over 50% of the human erythrocyte beta-spectrin subunit, were isolated by antibody screening of a lambda gt11 expression library constructed from human fetal liver mRNA. The amino acid sequence of the C-terminus of beta-spectrin was derived. The size of beta-spectrin mRNA in human erythroleukemia cells was found to be 7.5 kb. Erythrocyte beta-spectrin is encoded by a gene located on human chromosome 14, as determined by cDNA hybridization to human X mouse somatic cell hybrids.

Amino Acid Sequence↗
PubMed1985

A possible role for antibodies against spectrin in the interaction between erythroblasts and macrophages in vitro.

The nature of serum factors which participate in the interaction in vitro between dimethylsulphoxide-induced Friend leukaemia erythroblasts (IFLE) and syngeneic mouse peritoneal macrophages was investigated. When heat-inactivated newborn calf serum (HI-NBCS) was depleted of IgG its activity to promote the association of neuraminidase-treated 59Fe-labelled IFLE (59Fe-IFLE) with macrophages was markedly reduced but could be restored by the addition of bovine IgG. Trypsin treatment of macrophages caused incomplete inhibition of their subsequent association with both untreated and neuraminidase-treated 59Fe-IFLE in the presence of HI-NBCS. When spectrin, the major red cell cytoskeleton protein, was added to HI-NBCS there was a dose-related inhibition of the association with macrophages of both untreated and neuraminidase-treated 59Fe-IFLE. Moreover a mouse monoclonal antibody against spectrin promoted the interaction of neuraminidase-treated 59Fe-IFLE with macrophages. Mouse sera which supported the association of neuraminidase-treated 59Fe-IFLE with macrophages were found to contain anti-spectrin antibodies. These results suggest that IgG antibodies mediate the interaction between erythroblasts and macrophages via trypsin-sensitive and trypsin-resistant receptors on the macrophage surface and that at least some of the antibodies show specificity for spectrin.

Animals↗
PubMed1985

Sp alpha I/65: a new variant of the alpha subunit of spectrin in hereditary elliptocytosis.

Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two-dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Adult↗
PubMed1984

Is spectrin a calmodulin-binding protein?

The binding of calmodulin to spectrin from human erythrocytes has been studied by affinity chromatography on sepharose-calmodulin column. The alpha and beta spectrin chains, dissociated in 6-7 M urea, both bound to the sepharose-calmodulin column, but with different affinities. Both chains were eluted together by EGTA. Binding sites for calmodulin are, therefore, present in both alpha and beta chains. However, intact purified spectrin dimers did not bind to the sepharose-calmodulin column, which renders a physiological role of calmodulin-binding to spectrin rather unlikely.

Calmodulin↗
PubMed1984

Calcium regulation of magnesium dependent phosphorylation of human erythrocyte ghost spectrin.

Phosphorylation of human erythrocyte ghost membrane proteins was found to be affected by micromolar calcium concentrations. Increasing Ca2+ concentration to 0.2 microM decreased spectrin (band 2) phosphorylation to 30 +/- 6% of control (to which no calcium was added). Decreasing calcium concentration by adding EGTA (0.2mM) to the standard membrane preparation increased spectrin phosphorylation to 575% control. This effect of Ca2+ was more pronounced at higher temperature. At 0 degree C, Ca2+ (0.05mM) had no effect on protein phosphorylation. Sodium fluoride like EGTA caused a four to five fold increase in phosphorylation. Pyrophosphate, a phosphoprotein phosphatase inhibator, had no effect. Once spectrin was phosphorylated in the presence of [gamma-32P]ATP the addition of Ca2+ or EGTA did not decrease or increase its phosphorylation. It is suggested that calcium regulates spectrin phosphorylation either by decreasing kinase activity or by decreasing substrate availability.

Calcium↗
PubMed1983

The role of spectrin-dependent ATPase in erythrocyte shape maintenance.

The paper reports on the relationship between spectrin-dependent ATPase and erythrocyte shape. It can be seen from various experiments that different treatment of erythrocyte membrane which brings about alteration in activity of spectrin-dependent ATPase, also changes erythrocyte shape. We suggest that spectrin-dependent ATPase is a large actomyosin-like protein complex with some characteristics of contractile protein which, under suitable conditions, causes a physiological tension (contraction) of the membrane. Energetically rich state of spectrin-dependent ATPase seems to be responsible for this phenomenon.

Actomyosin↗
PubMed1984

Proteins closely related to spectrin and ankyrin are general components of cell membranes.

Membrane-associated analogues of erythrocyte spectrin and ankyrin have been detected in nonerythroid cells by crossreaction with antibodies. Brain spectrin and a spectrin-binding domain of brain ankyrin have been purified and demonstrated to have all known functional activities of their erythrocyte counterparts. The nonerythroid forms of spectrin and ankyrin may have functions such as providing support for the lipid bilayer, mediating some types of actin-membrane interactions, and integrating membrane-spanning proteins with cytoplasmic structural proteins.

Actins↗
PubMed1978

The spectrin phosphorylation reaction in human erythrocytes.

Phosphorylation of spectrin in whole cells occurs on a single tryptic peptide and indicates a specific and limited modification of spectrin. Phosphorylation of spectrin in soluble extracts and isolated membranes is less specific. Diamide is an inhibitor of erythrocyte spectrin kinase and casein kinase. This agent also inhibits the restoration of biconcave morphology to metabolically depleted erythrocytes reincubated with adenosine.

Adenosine Triphosphate↗
PubMed1983

Sulfhydryl reagents induce altered spectrin self-association, skeletal instability, and increased thermal sensitivity of red cells.

Incubation of erythrocytes with the sulfhydryl reagent N-ethyl-maleimide (NEM) results in altered spectrin self-association and formation of dimers on the membrane. Skeletons isolated from these cells exhibit marked skeletal instability. In addition, NEM treatment induces increased thermal sensitivity of both cells and purified spectrin. These effects were not produced in aerobically incubated glucose-6-phosphate dehydrogenase deficient cells and were therefore presumably not due to depletion of intracellular reduced glutathione. These effects were produced by another permeant sulfhydryl reagent, monobromobimane, but not by its membrane-impermeant derivative. We conclude that spectrin sulfhydryl groups play an important role in spectrin self-association and thermal stability.

Bridged Bicyclo Compounds↗
PubMed1980

In vivo and in vitro turnover of spectrin phosphate in erythrocytes.

Erythrocyte deformability may be modulated by regulatory phosphorylation of spectrin. Most investigators have assumed that the phosphate attached to spectrin turns over in vivo. We tested that assumption using dog erythrocytes. Erythrocytes were labeled in vitro with solutions containing 32P. After 1 week at 4 degrees C, 60% of the 32P had been incorporated into the erythrocytes, the erythrocytic gamma-32P-ATP had a specific activity of 0.285 muCi/mumol (S.E. = 0.0138), and a membrane extract enriched in spectrin had 15,200 +/- 2390 cpm/mg of protein. When these erythrocytes were labeled with 51Cr, washed with saline, and infused autologously, they had a normal lifespan (t1/2 = 24.9 +/- 1.24 days). The gamma-32P-ATP disappeared exponentially, with a t1/2 of 7.84 +/- 0.506 hr. The radioactivity in low-ionic-strength membrane extracts and Triton X-100 pellets declined exponentially, with a t1/2 of 33.4 +/- 0.336 and 39.1 +/- 0.806 hr, respectively. When 32P-labeled erythrocytes were incubated in vitro in a 15 mM phosphate buffer, the gamma-32P-ATP (t1/2 = 3.97 hr) and the radioactivity of low-ionic-strength extracts (t1/2 = 4.18 hr) and Triton pellets (t1/2 = 4.54 hr) declined rapidly. These rates of spectrin phosphorylation-dephosphorylation suggest that this process may be physiologically important and that in vitro turnover rates are significantly higher than those observed in vivo.

Animals↗
PubMed1982

Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes.

In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.

Anemia, Hemolytic↗
PubMed1982

A structural model of human erythrocyte spectrin. Alignment of chemical and functional domains.

Proteolytic susceptibility has been used to probe the structure of human erythrocyte spectrin. Nine unique polypeptide segments have been defined by mild trypsin digestion (0 degrees C) and analyzed by two-dimensional peptide mapping techniques. These peptide segments, referred to operationally as chemical domains, exhibited varying degrees of sensitivity to further proteolytic cleavage. One region (beta I) which contained the phosphorylated amino acids of the beta subunit was quite sensitive to proteolysis and was rapidly degraded to numerous small peptides. Overlap peptides produced by enzymatic and chemical cleavages were used to align each domain in the appropriate spectrin subunit. The molecular weights of the largest unique peptides from both subunits sum to the approximate weight of the intact molecule. Similarly, summation of the two-dimensional peptide maps of the intermediate sized peptides approximates the two-dimensional maps of the intact spectrin subunits, indicating that most or all of the molecule is represented. These results suggest that spectrin is composed of multiple, ordered, largely alpha-helical domains that are connected by small protease-sensitive segments. A comprehensive structural model is presented.

Electrophoresis, Cellulose Acetate↗