The erythrocyte membrane skeleton: biochemistry.
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Binding Sites↗
Biomedical subjects
S B Shohet
Publications and source records attributed to S B Shohet.
The erythrocyte membrane skeleton: pathophysiology.
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Anemia, Hemolytic, Congenital↗
Normal content of brain spectrin-like protein in sph/sph mice.
In the erythrocytes of WBB6F1-sph/sph mice spectrin constitutes only approximately 1% of the total sph/sph membrane protein compared to approximately 23% in WBB6F1-+/+ controls. No increase in proteolytic degradation of spectrin in sph/sph erythrocyte membranes could be detected with antibodies directed against mouse erythrocyte spectrin or mouse brain spectrin-like protein. As attachment of normal spectrin to the erythrocyte membrane of these animals appeared to be normal, and as spectrin is not detected when whole sph/sph erythrocytes are solubilized in SDS for SDS PAGE, the deficient erythrocyte spectrin was probably due to diminished production. Brain spectrin-like protein, a nonerythroid spectrin analogue, is antigenically, morphologically and functionally related to erythrocyte spectrin, but appears by peptide mapping analysis to be a distinct gene product. It was found by protein- and antibody-staining of brain membranes to be present in normal concentrations in sph/sph animals. Indirect immunofluorescence of mouse brain tissue with anti-brain spectrin-like protein IgG or anti-erythrocyte spectrin IgG indicated that the distribution of brain spectrin-like protein was normal in sph/sph brain. Therefore the mutation causing diminished production of sph/sph erythrocyte spectrin does not affect the expression of this nonerythroid spectrin analogue.
Animals↗
A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit.
A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.
Animals↗
Red cell membrane and cation deficiency in Rh null syndrome.
A 52-yr-old multiparous white female was found to have Rh null blood type. She had macrocytic anemia, with reticulocytosis (15%-20%), of long duration. Although stomatocytes in peripheral blood were numerous and osmotic fragility was increased, suggesting increased cell water, the RBC cation content, and thus cell water, was decreased. Cell dehydration was confirmed by an increased proportion of high density RBC on Stractan density gradients. The deformability of RBC from four gradient subpopulations was measured in the ektacytometer as a function of suspending medium osmolality. Analysis of these measurements showed an abnormal reduction in cell surface area with increasing cell density, thus explaining the increased osmotic fragility of whole blood. This was confirmed by a density-dependent reduction in cell cholesterol content, suggesting membrane instability in vivo. Rh null subpopulations showed a twofold increase in both ouabain-sensitive and -insensitive Na-K ATPase activity and 86Rb transport, even in the dense fraction with the fewest reticulocytes. No membrane protein or glycoprotein abnormality was detected by SDS-PAGE. The associated deficiencies of both membrane surface area and cation content in Rh null cells, as well as increased Na-K pump activity, suggest a pleiotropic functional interrelationship among Rh antigen, membrane stability, and cation regulation.
Anemia, Macrocytic↗
A novel phospholipid in irreversibly sickled cells: evidence for in vivo peroxidative membrane damage in sickle cell disease.
In individuals with sickle cell disease, a variable number of irreversibly sickled cells (ISC) is present that may contribute to the pathophysiology of sickle cell anemia. The present study was undertaken to determine the possible role of membrane lipid peroxidation in the genesis of ISC. After 24 hr of simple aerobic incubation, sickle cells accumulated 2-3 times more malonyldialdehyde (MDA), an end product of lipid peroxidation, than normal cells. To assess the possibility of peroxidative damage in ISC in vivo, ISC were separated from sickle blood using Stractan density gradients. Lipid extracts of the untreated ISC-enriched fraction of sickle blood showed significant fluorescence and contained a novel phospholipid:MDA adduct that was not seen in control cells. Taken together, these observations suggest that ISC have previously undergone lipid peroxidative damage and the accumulation of MDA in vivo.
Anemia, Sickle Cell↗
Polyamines do not inhibit erythrocyte ATPase activities.
To test whether physiologic elevation of red cell polyamine levels might explain Na pump inhibition in sickle cells or uremic red cells, we have studied the effect of putrescine, spermidine and spermine on red cell membrane ATPase and Na-K active transport. Measurement of the ouabain-sensitive influx of 86Rb into intact cells showed no effect of spermine. However, cells became depleted of ATP during incubation with spermine. By 48 h, the cells showed substantial potassium loss and moderate sodium gain. Because the low permeability of red cell membranes for polyamines might have obscured some direct effects on intracellular processes, we measured active transport of 22Na out of red cell ghosts that had been resealed in the presence of 5 mmol/l spermine. In addition, we measured the Na-K, Mg, and Ca ATPase activities of broken membrane preparations in the presence of spermine, spermidine and putrescine. Polyamines had no direct effect on cation transport in red cells, although possible adverse effects on red cell metabolism could have a secondary effect on cation regulation.
Adenosine Triphosphatases↗
Stabilization of erythrocyte membranes by polyamines.
Using a laser diffraction technique, we have studied the effects of putrescine, spermidine, and spermine, the three physiologic polyamines, on the deformability and mechanical stability of human erythrocyte membranes. Ghosts resealed with polyamines were subjected to high fluid shear stress in an ektacytometer. All polyamines increased the membrane shear modulus (decreased deformability) in a concentration- and time-dependent manner. The order of effectiveness was spermine greater than spermidine greater than putrescine. At 10 microM, spermine appreciably decreased membrane deformability. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytometer and deformability was continuously recorded as the deformable ghosts fragmented into rigid spherical vesicles. Polyamines, especially spermine, caused a noticeable increase in the t1/2 for fragmentation. These effects could not be ascribed to proteolysis or Ca2+-induced transglutamination. That the effects of polyamines were specific and not simply due to their positive charge was demonstrated by the finding that Ca2+ and Mg2+ destabilized the erythrocyte membrane as evidenced by decreasing the t1/2 for fragmentation. Extracellular polyamines were not effective except under conditions that caused significant accumulation inside the cell. The data indicate that intracellular physiologic polyamines, especially spermine, decrease erythrocyte membrane deformability and stabilize the membrane skeleton, making it more resistant to fragmentation.
Cell Membrane Permeability↗
Evidence of peroxidative damage to the erythrocyte membrane in iron deficiency.
The mechanism responsible for reduced red blood cell (RBC) survival in iron deficient infants or animals is unknown. To investigate the possible role of membrane peroxidation in iron-deficiency anemia, we studied RBC membrane lipids and proteins of rats fed iron-deficient (2 ppm Fe) and control (50 ppm Fe) diets between 21 and 41 days of age. Thin-layer chromatography of lipids showed that iron-deficient rats' RBC contained a novel phospholipid (1.9% of the total phospholipid) which moved between phosphatidylserine and phosphatidylethanolamine. Detailed studies showed that this PL is a Shiff's base adduct of phosphatidylserine, phosphatidyl-ethanolamine, and malonyldialdehyde, an end product of lipid peroxidation. Polyacrylamide gel electrophoresis of RBC proteins of iron-deficient rats also showed presence of high molecular protein complexes similar to that formed in in vitro malonyldialdehyde-treated RBC. To examine the role of such membrane cross-linking on in vivo RBC survival, we have studied survival of in vitro malonyldialdehyde-treated RBC in rabbits, 51Cr-T 1/2 of 5 microM malonyldialdehyde-treated RBC, which contained about the same amount of phospholipid/malonyldialdehyde adducts, was reduced to 6 days as compared to 11 days of sham-treated RBC. The in vitro study suggests that peroxidative damage results in significant reduction in RBC T 1/2 and may be analogous to decreased RBC survival in iron-deficient infants and animals.
Anemia, Hypochromic↗
The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr-survival of erythrocytes.
Erythrocyte membrane lipid peroxidation has been reported to occur in various haemolytic anaemias. In the present study, treatment of human erythrocytes with malonyldialdehyde (MDA), a product of fatty acid peroxidation, induced membrane rigidity, cellular dehydration and reduced whole cell deformability. These effects of MDA were blocked by histamine and fluorescamine, which can act as alternate substrates for MDA. Additionally, reduced deformability of MDA-treated rabbit cells was associated with shortened 51Cr survival in vivo. These findings suggest a biochemical basis for decreased survival of erythrocytes undergoing peroxidative damage of the membrane.
Animals↗
Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.
The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.
Adult↗
The influence of membrane skeleton on red cell deformability, membrane material properties, and shape.
A membrane skeleton consisting of a structural matrix of spectrin, actin, and band 4.1 linked to band 3 in the fluid bilayer through ankyrin appears to be responsible for many of the material properties of the red cell membrane. In response to externally applied forces, the membrane behaves as a solid, a semisolid, or a liquid, depending on the magnitude and duration of the applied forces. Under physiologic conditions, the normal skeleton permits the red cells to undergo marked reversible deformations as a viscoelastic material. Perturbations of this skeletal assembly, as a result of molecular defects in skeletal components, lead to various altered membrane material properties and altered behavior in the circulation. The altered material properties include increased elastic shear modulus, irreversible membrane flow, or even membrane yield, resulting in cell fragmentation. These alterations in turn lead to changes in cellular deformability either as a result of increased membrane rigidity or decreased surface-area-to-volume ratio, secondary to cell fragmentation. As cellular deformability is one of the major parameters that determines red cell life span, skeletal dysfunction leading to decreases in deformability can account for increased red cell destruction in many congenital and hereditary hemolytic anemias.
Anemia, Sickle Cell↗
Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance.
Whole cell deformability of red cells was measured as a continuous function of suspending medium osmolality using the ektacytometer, a laser-diffraction viscometer. Study of normal cells in which water content and membrane surface area had been selectively modified showed that this technique can detect changes in these properties with high sensitivity. The osmotic deformability profiles obtained from this assay provide information about cell water content, surface area, and the heterogeneity in these cellular properties, information that by conventional methods would require several different types of measurements. Application of this approach to a variety of pathologic blood samples showed that various hematologic disorders can be characterized by the shape of this profile and the position of specific features of the profile along the osmolality axis. Measurement of osmotic deformability profiles thus provides a convenient and comprehensive means of identifying abnormalities either in red cell water content or surface area.
Densitometry↗
Hereditary lecithin-cholesterol acyltransferase deficiency. Report of 2 new cases and review of the literature.
Two new cases of hereditary lecithin-cholesterol acyltransferase (LCAT) deficiency in a brother and sister born to consanguinous parents are reported. Both have corneal opacity, splenomegaly and mild hemolytic anemia. The brother, the older of the 2, also has significant proteinuria. The literature dealing with reported cases of hereditary LCAT deficiency and the clinical, pathological, diagnostic and management aspects of the disorder are reviewed.
Adult↗
Red cell membrane stiffness in iron deficiency.
The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron-deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.
Anemia, Hypochromic↗
Deformability of isolated red blood cell membranes.
We have used a laser diffraction method (ektacytometry) to directly measure the membrane component of red cell deformability, without contributions from either cell geometry or internal viscosity. This technique was validated by subjecting resealed erythrocyte ghosts to manipulations previously shown to increase the membrane shear modulus. Heating above 45 degrees C, pH greater than 9.0 and less than 5.0, and micromolar concentrations of the cross-linking agents, glutaraldehyde and diamide, all reduced the deformability of resealed erythrocyte ghosts. We have applied this assay to the study of reduced cellular deformability of calcium-loaded red cells, and have shown that, for physiological concentrations of calcium, the effect of calcium on the physical properties of the membrane may be negligible when compared to its effect of promoting cell dehydration and subsequent increased cytoplasmic viscosity.
Calcimycin↗
Lipid translocation across the human erythrocyte membrane. Regulatory factors.
A simple method based on the differential extraction of lysophosphatidylcholine (LPC) by saline and albumin solutions has been developed to study the factors that influence lipid translocation across intact human erythrocyte membrane. With this assay, the rate of LPC translocation across the bilayer at 37 degrees C was found to be 1.87% h (0.0187 h-1). Identical translocation rates were derived for normal cells and cells in which the ATP was totally depleted, implying that the metabolic state of the cell had no influence. In contrast, the translocation rate was strongly influenced by temperature. Above 21 degrees C, the rate doubled for every 51 degrees C increase in temperature, suggesting an important role for diffusion through the lipid phase. Denaturation of a single major skeletal protein, spectrin, by heating cells to 5 degrees C did not alter the translocation rate. However, oxidative cross-linking of a complex of membrane proteins by treatment with diamide significantly increased the rate of translocation at 37 degrees C. Cholesterol enrichment of the cells decreased the apparent rate of translocation but not the total quantity of LPC translocated. Taken together, these data suggest that lipid translocation across the intact human erythrocyte membrane is not energy dependent, and that it is influenced by the organizational state of both the lipid and protein moieties of the membrane.
Biological Transport↗
Red blood cell [14C]cholesterol exchange and plasma cholesterol esterifying activity of normal and sickle cell blood.
The present study performed on density fractions of sickle and normal erythrocytes prepared on Stractan density gradient shows that dense erythrocytes have consistently decreased uptake of [14C]cholesterol from plasma in comparison to young, less dense erythrocytes. Plasma of sickle cell patients also shows a reduction in cholesterol-esterifying activity in comparison to normal controls. A possible effect of these processes in the increased cholesterol to phospholipid molar ratio of irreversibly sickled cells has been suggested.
Anemia, Sickle Cell↗