scholarly journals A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1006-1015 ◽  
Author(s):  
M Garbarz ◽  
D Dhermy ◽  
MC Lecomte ◽  
C Feo ◽  
I Chaveroche ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Membranes ◽  
Red Cell ◽  
Intact Cells ◽  
Additional Band ◽  
Whole Cells ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Sds Page

Abstract A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

scholarly journals A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1006-1015
Author(s):  
M Garbarz ◽  
D Dhermy ◽  
MC Lecomte ◽  
C Feo ◽  
I Chaveroche ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Membranes ◽  
Red Cell ◽  
Intact Cells ◽  
Additional Band ◽  
Whole Cells ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Sds Page

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

scholarly journals Hereditary elliptocytosis with protein band 4.1 deficiency in the dog

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 373-377 ◽  
Author(s):  
JE Smith ◽  
K Moore ◽  
M Arens ◽  
GA Rinderknecht ◽  
A Ledet
Keyword(s):  
Animal Model ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Deformability ◽  
Membrane Stability ◽  
Osmotic Sensitivity ◽  
Hereditary Elliptocytosis ◽  
Membrane Cytoskeleton ◽  
Glutathione Deficiency

Abstract A dog with persistent elliptocytosis was studied. The dog had membrane protein band 4.1 deficiency, microcytosis, shortened erythrocyte lifespan, increased osmotic sensitivity, and a mild glutathione deficiency. Erythrocyte deformability and membrane stability were adversely effected. The dog's parents had decreased band 4.1, decreased stability, and some elliptocytosis. This disorder in dogs closely resembles human patients with band 4.1 deficiency and should provide a valuable animal model to study the erythrocyte membrane cytoskeleton.

scholarly journals Effect of membrane splitting on transmembrane polypeptides.

1986 ◽  
Vol 102 (2) ◽  
pp. 551-559 ◽  
Author(s):  
K A Fisher ◽  
K C Yanagimoto
Keyword(s):  
Erythrocyte Membrane ◽  
Periodic Acid ◽  
Freeze Fracture ◽  
Anion Channel ◽  
Band 3 ◽  
Intact Cells ◽  
Coomassie Blue ◽  
Sds Page ◽  
Before And After ◽  
Polypeptide Backbone

We investigated the effect of membrane splitting on the primary structure of human erythrocyte membrane polypeptides. Monolayers of intact, chemically unmodified cells were freeze-fractured and examined by one-dimensional SDS PAGE. Silver-stained gels revealed all major polypeptides that stain with Coomassie Blue as well as all bands that stain with periodic acid Schiff's reagent. Both nonglycosylated and glycosylated membrane polypeptides could be detected at concentrations of only a few nanograms per band. Membrane splitting had no effect on the position or number of bands. Monolayers of intact erythrocytes that had been enzymatically radioiodinated with lactoperoxidase were examined by electrophoresis, fluorography, and liquid scintillation counting. Radioactivity was quantified before and after monolayer formation and splitting, and at several stages of gel staining, drying, and fluorography. Although overexposed fluorographs revealed several minor radioiodinated bands in addition to band 3 and the glycophorins, no new bands were detected in split membrane samples derived from intact cells. These observations support the conclusion that neither the band 3 anion channel nor the glycophorin sialoglycoproteins are fragmented during freeze-fracturing. Although both band 3 and glycophorin partition to the cytoplasmic side of the membrane, preliminary quantitative observations suggest an enrichment of glycophorin in the split extracellular "half" membrane. We conclude that the process of membrane splitting by planar monolayer freeze-fracture does not cleave the covalent polypeptide backbone of any erythrocyte membrane protein, peripheral or integral.

scholarly journals Hereditary elliptocytosis with protein band 4.1 deficiency in the dog

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 373-377
Author(s):  
JE Smith ◽  
K Moore ◽  
M Arens ◽  
GA Rinderknecht ◽  
A Ledet
Keyword(s):  
Animal Model ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Deformability ◽  
Membrane Stability ◽  
Osmotic Sensitivity ◽  
Hereditary Elliptocytosis ◽  
Membrane Cytoskeleton ◽  
Glutathione Deficiency

A dog with persistent elliptocytosis was studied. The dog had membrane protein band 4.1 deficiency, microcytosis, shortened erythrocyte lifespan, increased osmotic sensitivity, and a mild glutathione deficiency. Erythrocyte deformability and membrane stability were adversely effected. The dog's parents had decreased band 4.1, decreased stability, and some elliptocytosis. This disorder in dogs closely resembles human patients with band 4.1 deficiency and should provide a valuable animal model to study the erythrocyte membrane cytoskeleton.

Characterization of Glycolytic Enzyme Complexes on Murine Erythrocyte Membranes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1571-1571
Author(s):  
Philip S. Low ◽  
Estela M. Campanella ◽  
William A. Anong ◽  
Nancy J. Wandersee ◽  
Cheryl A. Hillary ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Erythrocyte Membrane ◽  
Band 3 ◽  
Glycolytic Enzyme ◽  
Glycolytic Enzymes ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Intact Cells ◽  
Anion Transporter ◽  
Enzyme Complexes

Abstract Glycolytic enzymes have been recently shown to exist as multi-enzyme complexes in association with the cytoplasmic domain of band 3 at the inner surface of the human erythrocyte membrane. Because several of the glycolytic enzyme binding sites have been mapped to sequences near the NH2-terminus of band 3 (DDYED and EEYED) that are not conserved in mice (EEVLE and EELEN), the question naturally arose whether the existence of glycolytic enzyme complexes on erythrocyte membranes might be only a product of recent evolution. To test this hypothesis, fresh murine erythrocytes were fixed and stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and carbonic anhydrase II (CA II was used as a control, since it binds to a distant site near the COOH-terminus of band 3). Importantly, analysis of intact murine erythrocytes by confocal microscopy demonstrated that all of the above enzymes are localized to the membrane in oxygenated cells. In contrast, upon deoxygenation of the intact cells, release of the glycolytic enzymes (but not CA II) from the erythrocyte membrane and their uniform redistribution throughout the cytoplasm is observed. Because deoxyhemoglobin has been shown in human erythrocytes to compete with glycolytic enzymes (but not with CA II) for a common binding site at the NH2-terminus of band 3, these data argue that murine band 3, despite its weak homology to human band 3, still constitutes an organization center for glycolytic enzymes on the erythrocyte membrane. To further test this hypothesis, erythrocytes from band 3 knockout mice were similarly examined by confocal microscopy. Not surprisingly, all of the enzymes in all of the cells were evenly distributed throughout the cytoplasm, regardless of the oxygenation state of the cell. Further, immunoblot analyses demonstrated that glycolytic enzyme content of the band 3 knockout erythrocytes was measurably reduced compared to healthy mice, suggesting that the anion transporter may also contribute to enzyme stabilization during the lifetime of the erythrocyte. Finally, to determine whether the integrity of other membrane structures might impact the assembly of glycolytic enzyme complexes on the erythrocyte membrane, α-spectrin deficient mice were also examined for their enzyme distributions. Curiously, > 50% of the cells in any field exhibited glycolytic enzyme staining throughout the cytoplasm, with the remainder showing mainly membrane staining. Conceivably, the stabiity of glycolytic enzyme complexes on the membrane may also depend on the integrity of the membrane skeleton. Taken together, these data argue that glycolytic enzymes assemble in an oxygenation-dependent manner into complexes on murine erythrocyte membranes and that the stability of these complexes depends on the presence of band 3 and to a lesser extent α-spectrin. Supported by NIH grant GM24417.

scholarly journals Effects of Vitamin A on the Erythrocyte Membrane Surface

Blood ◽  
1973 ◽  
Vol 41 (6) ◽  
pp. 893-899 ◽  
Author(s):  
Martin J. Murphy
Keyword(s):  
Electron Microscope ◽  
Vitamin A ◽  
Scanning Electron Microscope ◽  
Erythrocyte Membrane ◽  
Membrane Surface ◽  
Erythrocyte Membranes ◽  
Red Cell ◽  
Cellular Events ◽  
Dose Dependent ◽  
Scanning Electron

Abstract The scanning electron microscope was used to examine the effects vitamin A has on the surface of human erythrocytes. The hemolytic properties of vitamin A, both time- and dose-dependent, are confirmed, and the observations are extended to the establishment of a sequence of cellular events that leads to the destruction of the red cell. The biochemical means whereby vitamin A induces these changes are discussed. In addition, the present study indicates that only selective areas of the erythrocyte membranes are responsive to the action of vitamin A, thereby supporting the proposal that the erythrocytic membrane may not be a homogeneous mass.

The Antiepileptic Drug Diphenylhydantoin Affects the Structure of the Human Erythrocyte Membrane

2004 ◽  
Vol 59 (5-6) ◽  
pp. 427-431 ◽  
Author(s):  
Mario Suwalsky ◽  
Sigrid Mennickent ◽  
Beryl Norris ◽  
Fernando Villena ◽  
Francisco Cuevas ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Cell Membranes ◽  
Erythrocyte Membranes ◽  
Repetitive Firing ◽  
Red Cell ◽  
Human Erythrocyte Membrane ◽  
Antiepileptic Agent ◽  
Red Cell Membrane ◽  
Spinal Cord Neurons

Phenytoin (diphenylhydantoin) is an antiepileptic agent effective against all types of partial and tonic-clonic seizures. Phenytoin limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord neurons maintained in vitro. This effect is mediated by a slowing of the rate of recovery of voltage activated Na+ channels from inactivation. For this reasons it was thought of interest to study the binding affinities of phenytoin with cell membranes and their perturbing effects upon membrane structures. The effects of phenytoin on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that phenytoin interacts with cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that phenytoin perturbed a class of lipids found in the outer moiety of cell membranes; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the erythrocyte membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the insertion of phenytoin in the outer monolayer of the red cell membrane. This is the first time that an effect of phenytoin on the red cell shape is described. However, the effects of the drug were observed at concentrations higher than those currently found in plasma when phenytoin is therapeutically administered.

Analysis of the red cell membrane in a family with hereditary elliptocytosis — total or partial of protein 4.1

1981 ◽  
Vol 59 (1) ◽  
pp. 68-71 ◽  
Author(s):  
Nicole Alloisio ◽  
Evelyne Dorléac ◽  
Robert Girot ◽  
Jean Delaunay
Keyword(s):  
Cell Membrane ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis

scholarly journals On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

1977 ◽  
Vol 73 (3) ◽  
pp. 647-659 ◽  
Author(s):  
W Birchmeier ◽  
SJ Singer
Keyword(s):  
Erythrocyte Membrane ◽  
Preceding Paper ◽  
State Level ◽  
Protein Molecule ◽  
Erythrocyte Membranes ◽  
Erythrocyte Ghosts ◽  
Intact Cells ◽  
Peptide Cleavage ◽  
Biochemical Effects ◽  
Shape Changes

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.

scholarly journals Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

1980 ◽  
Vol 86 (2) ◽  
pp. 694-698 ◽  
Author(s):  
C M Cohen ◽  
S F Foley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Actin Filaments ◽  
Binding Capacity ◽  
Protein Band ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Cytoplasmic Surface ◽  
Independent Association ◽  
Inside Out

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

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