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PubMed1979

Activation of Mg-ATPase (spectrin-dependent ATPase) by Ca2+.

The dependence of saponin-stimulated Mg-ATPase activity in the erythrocyte membrane on Ca2+ concentration was studied. In the membrane of freshly sampled human erythrocytes we found for this enzyme and Ca2+ an apparent dissociation constant of 0.611 mumol/l (SE +/- 0.106 mumol/l) and Hill coefficient of 0.93 (SE +/- 0.05). The enzyme is in most probability identical with Ca,Mg-ATPase of high affinity to Ca2+ described also as spectrin-dependent Ca,Mg-ATPase.

Adenosine Triphosphatases↗
PubMed1991

[The effect of supplying rats with vitamin K and administration of pelentane on Na,K- and spectrin-dependent ATPase in erythrocyte ghosts].

It was found that the activity of spectrin-dependent ATPase of erythrocyte ghosts isolated from rats with alimentary deficiency of vitamin K was significantly increased as compared with control animals, whereas in rats kept on a vicasol-rich diet this parameter was unchanged. In vitamin K-deficient rats the amount of proteins loosely bound to erythrocyte membranes was significantly reduced. At the same time, the activity of the integral enzyme (Na, K-ATPase) did not depend on the vitamin K provision despite the fact that in vitamin K-deficient animals kept on a vicasol-rich diet the enzyme affinity for ouabain was strongly decreased as compared with control. It was suggested that this effect might be due to the changes in the lipid and protein environment of the membrane-bound enzyme. Administration of the antivitamin K, pelentane, did not induce any conspicuous changes in the enzyme activities. It was concluded that antivitamin K does not induce any modification of the properties of erythrocyte-linked enzymes observed under conditions of vitamin K deficiency.

Adenosine Triphosphatases↗
PubMed1990

The cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240-kDa protein exhibiting immunochemical cross-reactivity with spectrin.

A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.

Animals↗
PubMed1990

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells.

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.

Animals↗
PubMed1990

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking.

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.

Diamide↗
PubMed1987

Is Ca2+ effect on passive K+ transport mediated by spectrin--dependent ATPase?

The aim of this report was to find which part of the membrane is responsible for the Ca2+ dependence of membrane permeability to K+. We found that the enzyme activity of large contractile complex of membrane proteins, the so called spectrin-dependent ATPase (sp-ATPase) increases at certain Ca2+ concentrations when K+ permeability decreases and vice versa. Ca2+ apparent dissociation constant for sp-ATPase is 6 X 10(-7) M which is the value corresponding to findings of Porzig and Stoffel (1978) for Ca2+ binding to membrane with low K+ permeability. Moreover, 20 chemically quite different substances which inhibit sp-ATPase activity simultaneously increase in the same concentrations K+ permeability and vice versa. No exception was found. The results show that low membrane permeability to K+ occurs at such conformation of sp-ATPase at which its enzyme activity may fully be manifested whereas at other conformations permeability to K+ increases. The conformation of sp-ATPase seems to affect gating mechanism of a respective channel for passive K+ transport through membrane.

Adenosine Triphosphatases↗
PubMed1989

Reaction of Se with SH groups in spectrin is involved in the stabilization of erythrocyte membrane skeleton.

Na2SeO3 supplementation in the dialysis medium could obviously prevent the dissociation of spectrin from the erythrocyte membranes. Such Se effect could be eliminated by pretreatment of erythrocyte membranes with a SH-blocking reagent, iodoacetamide(IAA) or addition of a SH-compound, dithio-threitol. The fluorescence intensity of erythrocyte membranes labelled with the fluorescent probe N-(3-pyrenyl)-maleimide decreased with increasing Na2SeO3 concentration used for pretreatment of ghosts. 31P-NMR spectra of erythrocyte membrane dialyzed in the presence or absence of Na2SeO3 concentration showed a difference in chemical shift anisotropy (delta sigma) between these two samples. These data suggest that the stabilization effect is based on changes in lipid-protein interaction and conformation of membrane skeletal components induced by reaction of their SH groups with Na2SeO3.

Chemical Phenomena↗
PubMed1987

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin.

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.

Actinin↗
PubMed1987

Plectin and IFAP-300K are homologous proteins binding to microtubule-associated proteins 1 and 2 and to the 240-kilodalton subunit of spectrin.

Structural and functional characteristics of plectin from intermediate filament preparations of rat glioma C6 cells were compared to those of the intermediate filament-associated protein of Mr = 300,000 (IFAP-300K) of baby hamster kidney cells (Yang, H.-S., Lieska, N., Goldman, A.E., and Goldman, R.D. (1985) J. Cell Biol. 100, 620-631). After radiolabeling and proteolytic digestion under varied conditions, both proteins yielded nearly identical peptide maps. Immunological cross-reactivity, co-migration on one- and two-dimensional high-resolution gels, chromatofocusing, and amino acid analysis demonstrated structural homology as well. In vivo labeling with 32Pi showed that plectin was the target for cAMP-independent protein kinases which phosphorylated 18-kDa domains at the end(s) of the molecule. Previously reported phosphorylation sites for cAMP-dependent and a newly identified site for Ca2+/calmodulin-dependent protein kinases were located on different domains. In solid-phase binding assays, plectin bound to vimentin, microtubule-associated proteins 1 and 2, the 240-kDa chain of brain fodrin, and alpha-spectrin from human erythrocytes. Similar characteristics were revealed for corresponding 300-kDa components of various other cell lines, supporting the concept that plectin is a general cytoskeletal cross-linking element, probably of multiple function.

Animals↗
PubMed1981

On the mechanism of red blood cell shape change and release of spectrin-free vesicles.

Changes in the negative change densities on the outside (by raising the phosphatidic acid concentration) or on the inside of the red blood cell membrane (by a breakdown of the polyphosphoinositides and a concomitant diacylglycerol production) do not explain the last step in echinocytosis, the release of spectrin-free vesicles.

Adenosine Triphosphate↗
PubMed1982

Monoclonal antibodies as probes of domain structure of the spectrin alpha subunit.

A library of nine monoclonal antibodies which bind to the alpha subunit of human erythrocyte spectrin has been established. The specificity of these antibodies confirms the alignment and uniqueness of each of the five previously identified peptide domains in this subunit and establishes the identity of additional smaller proteolytic peptide fragments. This immunochemical approach is complementary to the identification of peptide relationships by two-dimensional chymotryptic peptide mapping, and the results of both methods are in complete agreement.

Antibodies, Monoclonal↗
PubMed1994

Annexin VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin).

Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with 125I-annexin VI using blots of SDS-polyacrylamide gels. The binding was Ca(2+)-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target proteins is a protein-protein interaction. One of annexin VI-binding proteins with M(r) 240,000 was enriched in the cytoskeletal fraction and was identified as calspectin (brain spectrin or fodrin). When the binding was examined with purified calspectin in the native state, the Ca2+ affinity (KCa) was 7.6 microM, and the affinity for annexin VI (Kd) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alpha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits striking homology with the NH2-terminal regions of dystrophin and alpha-actinin. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca2+/PS-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin in the presence of Ca2+ and PS. These results suggest that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane and that annexin VI may be involved in the regulation of the membrane skeleton of neuronal cells in response to Ca2+.

Actins↗
PubMed1993

Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain.

Isoforms of ankyrin (ankyrinR) are expressed in both the erythrocyte and the brain. Four cDNAs representing regulatory domains of ankyrinR expressed in the rat spleen and brain were cloned and sequenced. These different cDNAs were found to result from tissue-specific alternative mRNA processing of the ankyrinR regulatory domain. One of the isolated cDNAs was used to develop an antibody to brain isoforms of ankyrinR, and this antibody was used to study the localization of ankyrinR in the rat brain. The protein was found to be widely expressed in neurons of the metencephalon but limited to a discrete subset of neurons in the rat forebrain. In the thalamus and areas of the basal ganglia, these neurons were grouped in defined nuclei, whereas in the cortex, hippocampus, and caudate putamen they appeared as isolated cells distributed randomly throughout these structures. A similar study using an antibody raised against erythrocyte spectrin (beta R) showed a comparable localization to that of ankyrinR. Both proteins were expressed late in the developing rat brain, as part of the maturation stage of neural development. These data suggest a specific role for these erythrocyte structural proteins in the postmitotic development of a subset of neurons in the rat brain.

Aging↗
PubMedDate not supplied

[A comparative study of the properties of ouabain-sensitive phosphatase in rat erythrocyte ghosts and spectrin-free membranes].

The removal of the membrane skeleton proteins (MSP), chiefly spectrin and actin, from the rat erythrocyte ghosts was shown to result in a decrease of both the total Na, K-ATPase activity and a partial reaction of the enzyme, namely the phosphatasic one. Besides, modulating effects of the effectors promoting the enzyme conformational transitions (ATP and Mg2+) on the ouabain-sensitive K-phosphatase is changed. For instance, a pronounced activation of the K-phosphate in a high-potassium medium in the presence of 1 mM ATP disappeared and the degree of the enzymatic activity enhancement in response to increasing MgCl2 concentrations (from 1.5 to 6 mM) is decreased. The data obtained are discussed from the viewpoint of possible involvement of the erythrocyte MSP in the catalytic of Na, K-ATPase.

Actins↗
PubMedDate not supplied

[Temperature denaturation of erythrocyte spectrin: rheology, deformability and resistance to detergents].

Detergent resistance of erythrocytes and viscosity of whole blood altered sharply already on the first stages of spectrin denaturation process (46-48 degrees C). Alternatively, deformability changes arise above 50 degrees C. Further insignificant increase in viscosity proceeded above 50 degrees C. Yield stress decreased to zero at t < 50 degrees C and at higher temperatures suspension demonstrated Newton's behavior. So, the following order of events can be seen: 1) the initial stages of denaturation process (46-48 degrees C) lead to shape modifications of erythrocytes (spicula formation on the cell rim-echinocytosis), with the detergent resistance of erythrocytes decreasing significantly and capability of echinocytes to aggregate with fibrinogen participation being lost. 2) Above 50 degrees C vesiculation and spherulation of erythrocytes lead to a sharp alteration of deformability with an insignificant increase of viscosity. Thus, deformability of erythrocytes, which is a very important factor in the capillary circulation, may be of lesser rheological role in large vessels.

Detergents↗
PubMed1987

Immunocytochemical localization of calspectin (a non-erythroid spectrin-like protein) in thyroid glands of normal and TSH-treated rats.

The localization of calspectin (fodrin, a non-erythroid spectrin-like protein), which is known to bind calmodulin and F-actin, was detected in the thyroid gland of normal and TSH-treated rats by means of light-microscopic immunocytochemistry. Calspectin was demonstrated in the cytoplasm of the follicle epithelial cells especially along the baso-lateral plasma membrane in normal rats. In TSH-treated animals, in addition to the baso-lateral plasma membrane region, the apical plasma membrane region of the follicle epithelial cells also showed positive reaction to the immunostaining. These results suggest that calspectin, in conjugation with calmodulin and actin, play a role in the secretory activities including reabsorption activity of colloid of the follicle epithelial cell.

Animals↗
PubMed2004

Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide.

BACKGROUND: Podocyte alpha-actinin-4 (actinin-4) is an essential component of the glomerular filtration barrier. We recently reported that the central rod spectrin-like repeats (R1-R4) of actinin-4 have a high affinity to puromycin aminonucleoside (PAN), which can induce nephro-sis in animals. The aim of this study was to identify endogenous molecules that interact with the actinin-4 R1-R4 domain. METHODS: To identify such molecules, we performed a bacterial two-hybrid screening of a human kidney cDNA library using as a bait human actinin-4 R1-R4. We further verified the identified interactions by in vitro affinity assays and immunofluorescent studies of cultured human embryonic kidney HEK293 cells. To investigate the expression of the identified molecules in podocytes, in situ hybridization, and immunohistochemical studies were performed. RESULTS: One isolated cDNA from the library encoded humanin, a recently identified antiapoptotic peptide. In vitro affinity assays showed specific interactions of recombinant actinin-4 R1-R4, R1, R2, R3, and R4 proteins with humanin-Sepharose. PAN had no effect on these interactions. Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells. Altered colocalization was not observed by the addition of PAN. In situ hybridization and immunohistochemistry showed the expression of humanin in podocytes. CONCLUSIONS: Our results suggest that humanin is a novel binding partner of the actinin-4 R1-R4 domain in podocytes. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.

Actinin↗
PubMed1975

Extraction and localization of a (Ca2+ and Mg2+)-stimulated ATPase in human erythrocyte spectrin.

(1) A water soluble (Ca2+ plus Mg2+)-activated APTase has been extracted with 0.1 mM EDTA and 0.1 mM ATP from human erythrocyte membranes. (2) The specific activity of the extracted protein is increased 4- to 6-fold in comparison with untreated ghosts. (3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [gamma-32P]ATP-labeled erythrocyte membranes shows that the (Ca2+ plus Mg2+)-activated ATPase is located in the "spectrin" region (Mr 220 000-240 000). The radioactivity of these high molecular peptide bands is decreased markedly after the extraction of the ATPase at low ionic strength.

Adenosine Triphosphatases↗