A molecular abnormality of the erythrocyte membrane in beta-thalassemia

We present a new abnormality of the red cell membrane observed in the course of a study on beta-thalassemia. In a child suffering from a beta 0-thalassemia major, the neutral membrane bound phosphatase, instead of having Michaelis-Menten kinetics, displayed a biphasic kinetics consistent with inhibition by substrate excess. This abnormality is discussed along with other erythrocyte membrane alterations that are known to occur in beta-thalassemia.     

Oligomeric states of spectrin in normal erythrocyte membranes: Biochemical and electron microscopic studies

We estimated the relative amounts of oligomeric species of spectrin in 0°C red-cell-membrane extracts, including those released from spectrin-actinpolypeptide 4.1 complexes after mild urea treatment. Spectrin dimers, tetramers, and medium-size oligomers were the prominent species, accounting for 5%–10%, 45%–55%, and 25%–35% of spectrin, respectively. When examined by low-angle rotaryshadowing electron microscopy, these medium-size spectrin oligomers (e.g., hexamers, octamers, decamers, dodecamers, and quadecamers) appeared as polyskelions formed by head-to-head association of three to seven dimers. They were stable species capable of binding to, and subsequent release from, inside-out vesicles without degradation to tetramers or dimers. The data suggest that spectrin tetramers and medium-size oligomers coexist in the normal erythrocyte membrane as the primary native spectrin species.     

Abnormal membrane protein methylation and merocyanine 540 fluorescence in sickle erythrocyte membranes

Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.     

Hydrogen-ion titration studies on erythrocyte membranes.

1. H+ titration was used to detect the presence of ionizable groups on human erythrocyte plasma membranes. Between pH2.9 and 11.3, two significant peaks of H+ association/dissociation occur in the differential from of the titration curve, one at pH3. 1. And the other at pH10.3. 2. After disruption of membrane structure by exposure to high pH or by the addition of sodium dodecyl sulphate, maxima of H+ association/dissociation were seen at pH3.1,4.3,6.5,10.3 and 10.7. 3. Spectrophotometric assay and selective chemical treatments were used to identify several of the titratable residues. 4. The degree of eleectrostatic interaction between titratable charged groups was investigated by comparing the titration characteristics of the membranes before and after modification of membrane structure.     

Effect of acute intestinal obstruction on erythrocyte membrane function

The amino acid composition of red blood cell membrane proteins had been studied in different stages of acute intestinal obstruction. Hydrophobic amino acids were revealed to increase and glutamate was found to decrease during the early period of acute intestinal obstruction. Later neutral amino acids and some of the main amino acids were stated to decrease. Shifts in the ratio of protein fractions seen in red blood cell membrane of rats with acute intestinal obstruction could be explained by changes followed in the amino acid composition. The data accumulated had demonstrated that such a significant modification of protein component of the red blood cell membrane could be one of the reasons of the erythrocyte membrane penetrability violation and could play the pathogenetic role in the occurrence of irreversibility changes in cases of the intestinal obstruction. All that was mentioned above had shown the necessity to use membrane protectors and antienzyme drugs in the postoperative period.     

Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.     

Biochemical profile of erythrocyte membrane of jaundiced neonates

Studies in newborn humans have demonstrated alteration in the lipid, phospholipid and cholesterol content when compared with age-matched control. Membrane bound (Na+ + K+)ATPase activity is found to be significantly increased in jaundiced neonates. Alteration in membrane permeability characteristics in jaundiced neonates causes severe microenvironmental changes in red blood cell profile.     

Fluorescence studies of the aged erythrocyte membranes

The most important purpose of this research is to characterize by means of fluorescence polarization the structural and functional changes which occur in the membrane of the human erythrocytes during aging process. Our results provide evidence of a significant increase of membrane fluidity in the deep lipid core and in the lipid/protein boundary, in the aged erythrocytes. These features are associated with a rigidity of the membrane surface, as revealed by the anisotropy increase of a specific probe suitable for monitoring the membrane protein behaviour. These modifications could be considered as one of the mechanisms which contribute to alter erythrocyte rheological properties sufficiently to be recognised and removed within circulation.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Kinetic studies of human erythrocyte membrane resealing

Following lysis in hypotonic media, human erythrocyte membranes will spontaneously reseal and regain their original low permeability for polar solutes. It is generally accepted that resealing will only occur when the membranes are heated above a critical temperature, and that the membrane lesions are stable under cold conditions. Contrary to these prevailing notions, a detailed investigation of the temperature dependence of resealing kinetics over the temperature range 0–22°C revealed that resealing occurs at measurable rates at temperatures as low as 0°C, even in buffers of low ionic strength. At all temperatures studied, initial resealing rates were approximately first-order, and Arrhenius plots of these rates revealed a sharp, singular discontinuity at approx. 7°C.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

The 'hollow cylinder' protein of erythrocyte membranes.

The 'hollow cylinder' protein (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534-537) has been purified from human erythrocyte membranes. The molecular weight of the native protein, as determined by analytical ultracentrifugation, was found to be 747,000. By means of sodium dodecyl sulphate gel electrophoresis, the purified protein was shown to be composed of three different low molecular weight polypeptides of average molecular weight 25,000. This study provides convincing evidence that the spectrin tetramer is not responsible for the characteristic electron microscopic appearance of the hollow cylinder protein.     

Water exchange through erythrocyte membranes: Biochemical and nuclear magnetic resonance studies re-evaluating the effects of sulfhydryl reagents and of proteolytic enzymes on human membranes

Summary The water permeability of human red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on intact cells and resealed ghosts following exposure to various sulfydryl-reacting (SH) reagents and proteolytic enzymes. The main conclusions are the following: (i) When appropriate conditions for exposure of erythrocytes or ghosts to mercury-containing SH reagents (concentration, temperature and duration of incubation) were found, the maximal inhibition of water diffusion could be obtained with all mercurials (including HgCl₂ and mersalyl that failed to show their inhibitory action on RBC water permeability in some investigations). While previous studies claimed that long incubation times are required for the development of maximal inhibition of water diffusion by mercurials, the present results show that it can be induced in a much shorter time (5–15 min at 37°C) if relatively high concentrations of PCMBS (2–4mm) are used and no washings of the inhibitor are performed after incubation. Higher than optimal concentrations of mercurials and/or longer incubation times result in lower values of inhibition, sometimes a loss of inhibition, or can even lead to higher values of permeability compared to control RBCs. (ii) The conditions for inhibition by mercurials are drastically changed by preincubation of erythrocytes with noninhibitory SH reagents (such as NEM or IAM) or by exposure to proteolytic enzymes. If the cells are digested with papain, the duration of incubation with PCMBS should be decreased in order for inhibition to occur. This explains the lack of inhibition reported previously, when a relatively long duration of incubation with PCMBS was used subsequent to papain digestion. (iii) The degree of inhibition of water diffusion induced by mercurials appeared to be dependent upon the temperature of which the water permeability was measured. The values of maximal inhibition ranged from 45–50% at 37°C, increased 10–15% at 20°C and further increased at lower temperatures, reaching values above 75% below 10°C; these results clarify the conflicting reports of various authors. (iv) The inhibition of water diffusion, either reversible, or irreversible, was not accompanied by significant changes in the pattern of RBC membrane polypeptides fractionated by polyacrylamide gel electrophoresis. (v) The mean value of the activation energy of water diffusion (E _a,d) obtained on 42 donors was 25.6 kJ/mol. The values ofE _a,d increased in parallel with the values of the inhibition of water diffusion induced by PCMBS until the maximal inhibition was reached (whenE _a,d=41 kJ/mol) and then both sets of values decreased in parallel.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and re-iodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with ¹²⁵I, formation of sealed inside-out vesicles and re-iodination with ¹³¹I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of ¹²⁵I to ¹³¹I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.