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 Shape and volume changes in erythrocyte ghosts and spectrin-actin networks.

1980 ◽  
Vol 86 (2) ◽  
pp. 371-376 ◽  
Author(s):  
R M Johnson ◽  
G Taylor ◽  
D B Meyer
Keyword(s):  
Erythrocyte Membrane ◽  
Cell Shape ◽  
Electrolyte Concentration ◽  
Control Cell ◽  
Erythrocyte Ghosts ◽  
Volume Changes ◽  
Sulfhydryl Reagents ◽  
Actin Networks ◽  
Energy Dependent ◽  
Shape Changes

In response to changes in electrolyte concentration and pH, erythrocyte ghosts can exhibit some of the characteristic shapes seen in the intact erythrocyte. These shape changes are accompanied by volume changes; both are reversible, not energy dependent, and not inhibited by sulfhydryl reagents. The volume reduction can also be seen in isolated Triton-free spectrin-actin lattices, showing that this network is capable of reversible contraction. The results suggest that reversible changes in size of the underlying cytoskeleton of the erythrocyte membrane can control cell shape.

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 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 Optical-rotatory-dispersion studies of compounds related to cholesterol in liposomes and the membranes of erythrocyte ‘ghosts’

1973 ◽  
Vol 135 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Jonathan R. Green ◽  
Peter A. Edwards ◽  
Colin Green
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Erythrocyte Membranes ◽  
Optical Rotatory ◽  
Erythrocyte Ghosts ◽  
Polar Solvents ◽  
Human Erythrocyte Membranes ◽  
Do So

1. Steroid molecules containing the α,β-unsaturated oxo group in various positions were incorporated with egg phosphatidylcholine into liposomes and into human erythrocyte membranes. 2. The liposomes formed contained 0.3–0.94mol of steroid/mol of phospholipid and the steroids replaced 19–76% of the erythrocyte membrane sterol. 3. The optical rotatory dispersion (o.r.d.) spectra of the steroids in these structures were compared with those obtained in solvents of different polarity. 4. The o.r.d. spectra of cholesta-4,6-dien-3-one and 3-hydroxycholest-3-en-2-one in liposomes resembled those obtained with polar solvents such as ethanol or triethyl phosphate–water (1:1, v/v). 5. The o.r.d. spectra of 3-hydroxycholest-7-en-6-one and 3-hydroxycholest-5-en-7-one in liposomes resembled those obtained with moderately polar solvents such as dioxan. 6. The o.r.d. spectrum of 3-hydroxycholest-8(14)-en-15-one in liposomes resembled those obtained with non-polar solvents such as cyclohexane. 7. 3-Hydroxycholest-3-en-2-one did not exchange with erythrocyte membrane cholesterol, but the other steroids did do so and the o.r.d. spectra of the membranes containing them closely resembled those obtained with liposomes. 8. From the results, the position of sterol molecules with respect to the phospholipid molecules in liposomes and membranes of human erythrocyte ‘ghosts’ can be deduced.

scholarly journals Lipid organization in erythrocyte membrane microvesicles

1984 ◽  
Vol 224 (1) ◽  
pp. 285-290 ◽  
Author(s):  
S Scott ◽  
S A Pendlebury ◽  
C Green
Keyword(s):  
Phospholipase A2 ◽  
Erythrocyte Membrane ◽  
Human Erythrocytes ◽  
The Other ◽  
Erythrocyte Membranes ◽  
Erythrocyte Ghosts ◽  
Lipid Organization

The aminophospholipids of microvesicles released from human erythrocytes on storage or prepared from erythrocyte ghosts by shearing under pressure are susceptible to the action of 2,4,6-trinitrobenzenesulphonic acid. The aminophospholipids of the former vesicles are also susceptible to attack by phospholipase A2. Under the same conditions, the aminophospholipids of erythrocytes undergo little reaction. This suggests that the phospholipids in microvesicle membranes are more randomly distributed than those in erythrocyte membranes. Measurements have also been made of the ability of filipin to react with the cholesterol of sealed and unsealed erythrocyte ghosts and of microvesicles prepared from them. From the initial rates of reaction, it was concluded that there is no preferential transfer of cholesterol molecules from one side of the bilayer to the other during the formation of the microvesicles.

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 On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex.

1977 ◽  
Vol 73 (3) ◽  
pp. 638-646 ◽  
Author(s):  
M P Sheetz ◽  
S J Singer
Keyword(s):  
Human Erythrocyte ◽  
Network Formation ◽  
Structural Effect ◽  
Erythrocyte Membranes ◽  
Cross Linking ◽  
Intact Cells ◽  
Cytoplasmic Face ◽  
Human Erythrocyte Membranes ◽  
Shape Changes

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.

Calcium-dependent hydrolyses of polyphosphoinositides in human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 363-368 ◽  
Author(s):  
R. Blaine Moore ◽  
Stanley H. Appel
Keyword(s):  
Time Course ◽  
Erythrocyte Membranes ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Calcium Activation ◽  
Activation Curve ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Human Erythrocyte Membranes ◽  
Phosphatidylinositol 4

Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 °C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 μM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca–EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.

scholarly journals Effect of Hyperglycemia on the Total Surface Charge of the Erythrocyte Membrane in Patients with Metabolic Syndrome

2019 ◽  
Vol 15 (3) ◽  
pp. 322-327
Author(s):  
V. I. Podzolkov ◽  
T. V. Koroleva ◽  
M. G. Kudryavtseva
Keyword(s):  
Metabolic Syndrome ◽  
Surface Charge ◽  
Erythrocyte Membrane ◽  
Negative Charge ◽  
Fasting Blood Glucose ◽  
Microvascular Blood Flow ◽  
Erythrocyte Membranes ◽  
Vascular Changes ◽  
Group 2 ◽  
Group 1

Aim. To study the effect of hyperglycemia on the total surface charge of the erythrocyte membrane (SCEM) in patients with metabolic syndrome (MS).Material and methods. 112 MS patients were examined (45 men and 67 women) (mean age 61.4±7.2 years, average MS duration 8.7±5.2 years). The level of SCEM was determined by adsorption of a positive cationic dye (cationic blue O) on the surface of the plasma membrane of erythrocytes to completely neutralize their negative charge, followed by photometry of the solution and calculation of the number of charges on the cell surface of erythrocytes.Results. In the main group of patients with MS, abdominal obesity was observed in 100% of patients, arterial hypertension – in 73%, hyperglycemia – in 75%, dyslipidemia – in 80%. The level of glycated hemoglobin (HbA1c) was determined in all patients with MS, which was 7.3±1.9%. Patients with MS were conditionally divided according to the level of HbA1c into 2 groups (group 1 – HbA1c from 6.6 to 7.8%, group 2 – more than 7.8%). In MS patients with hyperglycemia, the SCEM values were significantly lower than in the group of patients without hyperglycemia (1.58±0.05×107 and 1.64±0.03×107, respectively; p=0.001)., Significant negative correlations between SCEM and the fasting blood glucose level, hyperglycemia duration, HbA1c level were found in patients with MS.Conclusion. SCEM indices reliably depended on the presence, severity and duration of hyperglycemia, which indicated the effect of impaired carbohydrate metabolism on the state of electric charge of erythrocyte membranes and, therefore, on the mechanisms of microvascular blood flow, thereby contributing to the development of vascular changes in patients with MS.

scholarly journals Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes

2000 ◽  
Vol 350 (2) ◽  
pp. 531-535 ◽  
Author(s):  
David W. C. DEKKERS ◽  
Paul COMFURIUS ◽  
Rein G. J. VAN GOOL ◽  
Edouard M. BEVERS ◽  
Robert F. A. ZWAAL
Keyword(s):  
Multidrug Resistance ◽  
Erythrocyte Membrane ◽  
Erythrocyte Membranes ◽  
Multidrug Resistance Protein ◽  
Multidrug Resistance Protein 1 ◽  
Lipid Asymmetry ◽  
Appreciable Change ◽  
Outer Leaflet ◽  
Resistance Protein ◽  
Phospholipid Distribution

The role of multidrug resistance protein 1 (MRP1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated. The transbilayer distribution of endogenous phospholipids and [(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRP1. At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids. Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx. 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25% and 3% for control cells respectively. To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet. No shift in phospholipid distribution was observed after 5h of incubation with verapamil or indomethacin. However, after 48h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet. No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine. Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy. This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.

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