Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Purification and characterization of two forms of a low-affinity Ca2+-ATPase from erythrocyte membranes

A low-affinity Ca²⁺-ATPase from erythrocyte membranes has been purified by agarose suspension electrophoresis and polyacrylamide gel electrophoresis in the absence of detergents. For maximal activity a calcium concentration above 10 mM is required. The activity is independent of magnesium. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP is about 60 μM. The enzyme appears in two forms (A and B) with similar amino acid composition. The specific activity of A is higher than that of B. Gel electrophoresis in SDS of A gives a pattern consisting of two bands. B gives the same pattern; the only difference between the patterns is the ratio of the amounts of protein in the bands. The apparent molecular weight of the proteins in the two SDS bands has been estimated at 23 000 and 21 000, respectively. The results obtained can be explained by assuming that the two proteins corresponding to the two bands obtained in SDS electrophoresis have a similar structure and can associate to complexes A and B. We have also shown that electrophoretic and chromatographic supporting media can induce aggregation of (membrane) proteins. Artificial complexes can thus be formed and cause misinterpretation of the data obtained. This may be the reason why some authors have speculated that Ca²⁺-ATPase is active only in complex with other proteins such as spectrin and actin.     

Echinophilic proteins stomatin, sorcin, and synexin locate outside gangliosideM1 (GM1) patches in the erythrocyte membrane

The detergent (Triton X-100, 4 °C)-resistant membrane (DRM)-associated membrane proteins stomatin, sorcin, and synexin (anexin VII) exposed on the cytoplasmic side of membrane were investigated for their lateral distribution in relation to induced ganglioside_M1 (GM1) raft patches in flat (discocytic) and curved (echinocytic) human erythrocyte membrane. In discocytes, no accumulation of stomatin, sorcin, and synexin in cholera toxin subunit B (CTB) plus anti-CTB-induced GM1 patches was detected by fluorescence microscopy. In echinocytes, stomatin, sorcin, and synexin showed a similar curvature-dependent lateral distribution as GM1 patches by accumulating to spiculae induced by ionophore A23187 plus calcium. Stomatin was partly and synexin and sorcin were fully recruited to the spiculae. However, the DRM-associated proteins only partially co-localized with GM1 and were frequently distributed into different spiculae than GM1. The study indicates that stomatin, sorcin, and synexin are echinophilic membrane components that mainly locate outside GM1 rafts in the human erythrocyte membrane. Echinophilicity is suggested to contribute to the DRM association of a membrane component in general.     

Permeability of human erythrocyte membrane vesicles to alkali cations

The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using ⁸⁶Rb as a K analog, we have measured the rate constant of ⁸⁶Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Isolation and characterization of mosquito cell membrane glycoproteins

Plasma membranes have been purified from an established cell line, Mos 20A of Aedes aegypti, and analysed for glycoprotein and polypeptide constituents by isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis. A major glycoprotein of molecular weight 110 000 carrying binding sites for concanavalin A and soybean agglutinin has been purified to homogeneity. Although located on the cell surface, the 110 kdalton glycoprotein is not labelled by lactoperoxidase-catalysed radioactive iodination of whole cells. Analysis indicates the presence of N-glycans, containing on average nine mannose residues, and the N-acetylglucosaminyl-β1,4-N-acetylglucosamine sequence. In addition, O-glycosidically linked N-acetylgalactosamine residues are present.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

Induced alteration of rat erythrocyte membrane with effect of pyrethroid based compounds

The effects of tetramethrin and prallethrin exposure on plasma total proteins, free amino acids, albumins, urea, urea nitrogen, uric acid, creatinine were tested. Serum SGOT, SGPT and lipid profile, antioxidants super oxide dismutase (SOD), catalase, GSH, G-Px, phospholipids, cholesterol, C/P ratio in membranes of erythrocyte and membrane fluidity were analyzed. The reason of the study were analyzed to examine the possessions of mosquito repellent pyrethroid (MRP) based compounds tetramethrin and prallethrin exposure on plasma profile, antioxidant status of erythrocyte membrane, membrane fluidity in male Wistar rats. We tested chronically for three months exposure every day (continuously for 8–10 h per day by inhalation) of tetramethrin and prallethrin markedly available (MRP) repellents treated on male Wistar rats. Our results confirmed that tetrarmethrin and prallethrin treatment effect of plasma profile alterations, and lipid homeostasis mechanism in Red Blood cells (RBCs). Tetramethrin and prallethrin treatment significantly increased in erythrocyte membrane phospholipids and decreased levels of cholesterol with no change of protein content, increased C/P ration levels. Inhalation of tetramethrin and prallethrin stimulate plasma biophysical and biochemical modify SGOT, SGPT, erythrocyte membrane cholesterol and phospholipid levels, individual phospholipids and membrane fluidity of exposure rats compared to controls.     

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction

Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with $\text{N-}\text{ethylmaleimide}$, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH₂ groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH₂ groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH₂ groups are not involved in the reaction.     

Characterization of the phosphorylated intermediate of the isolated high-affinity (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes

Phosphorylation of solubilized and purified high-affinity (Ca²⁺ + Mg²⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human erythrocyte membranes shows no dependence on cyclic AMP concentration in the range 0.1–1000 μM.Ca²⁺-dependent phosphoprotein is sensitive to hydroxylamine and molybdate treatment. The phosphate linkage shows maximum stability at low pH values, which is progressively lost as the pH rises, with a shoulder around pH 6. SDS gel electrophoresis of the phosphorylated protein yields a peak which shows relative mobility corresponding to a molecular weight of 145 000 and sensitivity to MgATP-chase and hydroxylamine treatment. This indicates that the phosphoprotein represents the phosphorylated intermediate of the high-affinity (Ca²⁺ + Mg²⁺)-ATPase of human erythrocyte membranes.     

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35–38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.     

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

X-ray diffraction and electron microscopy studies of frozen erythrocyte membrane preparations

Well-defined X-ray diffraction patterns have been recorded from erythrocyte membranes in the frozen state. At ?40°C, lamellar periodicities range from 19 to 95 nm depending on the glycerol content (0–40%, respectively). Freeze-fracture electron micrographs of samples frozen in two stages to approximate to the diffraction conditions show ice formation external to membrane stacks. The membrane stacks have periodicities of the same order of magnitude as those obtained by X-ray diffraction.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Enhanced activation of human erythrocyte Ca2+-ATPase by calmodulin after storage or brief exposure to disulfite

Ca²⁺-ATPase of human erythrocyte membranes which are prepared from freshly drawn human blood can be activated by the calmodulin present in the hemolysate to 1.5-times the basal level. However, when the membranes are prepared from blood stored for 5–14 days the activation by calmodulin reaches 2.5-times the basal level. An enhanced reactivity to calmodulin of similar magnitude was produced by brief exposure of fresh erythrocytes to 25 mM Na₂S₂O₅ prior to isolation of the membranes. Reincubation of the activated cells in a disulfite-free medium restored the membrane-bound Ca²⁺-ATPase to a state of normal reactivity to calmodulin. It is hypothesized that these results are related to the level of cytoplasmic Ca²⁺ which is partly controlled by complex formation with 2,3-diphosphoglycerate, the concentration of which is diminished when its specific phosphatase is activated by Na₂S₂O₅.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [³H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.     

The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5′-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg²⁺ or Mn²⁺. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5′-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.