Large scale isolation of human erythrocyte membranes by high volume molecular filtration

A molecular filtration procedure for preparing large quantities of human erythrocyte ghost membranes is presented. Hemolysate ghost membranes are rapidly cycled in the retantate channel of the filtration apparatus, while hemoglobin is removed s it pass through Pellicon filters into the filtrate. Several-liter quantities of washed packed erythrocytes can be processed in a few hours with this system and the filtration procedure does not appear to alter erythrocyte or ghost membranes. Intact erythrocytes in isotonic solution can be circulated through the retentate channel for 16 h with only 3% hemolysis and with preservation of their orginal morphology in scanning electron microscopy. Ghost membranes isolated by the procedure are virtually identical in morphology, polypeptide composition and acetylcholinesterase content to membranes isolated by conventional centrifugation techniques.     

Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes

Erythrocyte plasma membranes were isolated from a homogeneous population of human or rabbit erythrocytes fractionated into classes representing young, middle-age and old age in vivo. Lipid analyses of human erythrocyte plasma membranes reveal a decrease of the cholesterol to phospholipid molar ratio, followed by a marked decrease in the activities of the membrane-bound enzymes (Na+,K+)-stimulated ATPase, acetylcholinesterase and NAD+ase from young to old age. Such changes were not observed between young and middle-age rabbit erythrocytes. Incubation of rabbit young erythrocytes with phosphatidylcholine vesicles (liposomes) to obtain partial depletion of their membrane cholesterol, indicated that cholesterol depletion causes a statistically significant decrease of the (Na+,K+)-stimulated ATPase and acetylcholinesterase activities, but the NAD+ase activity remained almost unchanged. The biological significance of these data are discussed in terms of the differences and modifications in the interaction of membrane-bound enzymes with membrane lipids during in vivo ageing of erythrocytes.     

Different behavior of ghost-linked acidic and neutral sialidases during human erythrocyte ageing

Acidic and neutral sialidases (pH optimum 4.7 and 7.2, respectively) were assayed on human circulating erythrocytes during ageing. The assays were performed on intact erythrocytes and resealed erythrocyte ghost membranes. From young to senescent erythrocytes the acidic sialidase featured a 2.7-fold and 2.5-fold decrease in specific activity when measured on intact cells or resealed ghost membranes, whereas the neutral sialidase a 5-fold and 7-fold increase, respectively.The Ca²⁺-loading procedure was employed to mimic the vesiculation process occurring during erythrocyte ageing. Under these conditions the released vesicles displayed an elevated content of acidic sialidase, almost completely linked through a glycan phosphoinositide (GPI) anchor but no neutral sialidase activity, that was completely retained by remnant erythrocytes together with almost all the starting content of sialoglycoconjugates. The loss with vesiculation of acidic sialidase with a concomitant relative increase of neutral sialidase was more marked in young than senescent erythrocytes.The data presented suggest that during ageing erythrocytes loose acidic sialidase, and get enriched in the neutral enzyme, the vesiculation process, possibly involving GPI-anchors-rich membrane microdomains, being likely responsible for these changes. The enhanced neutral sialidase activity might account for the sialic acid loss occurring during erythrocyte ageing.     

Plasma membrane nadh dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.     

Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

Hypoxanthine phosphoribosyltransferase and hypoxanthine uptake in human erythrocytes.

A system of hypoxanthine uptake and IMP retention was studied and characterized in human erythrocytes. It follows closely the system already described for rabbit erythrocytes[7]. IMP formation and retention are dependent on the activity of hypoxanthine phosphoribosyl-transferase and on intracellular availability of phosphoribosyl pyrophosphate (P-Rib-PP), which is one of the substrates. In the extrecellular medium, neither P-Rib-PP nor GMP -- a potent inhibitor of the enzyme in vitro -- has any influence on IMP retention. The amount of residual hypoxanthine phosphoribosyltransferase in erythrocyte ghost preparations is directly related to the residual hemoglobin content. Thus the enzyme is characterized as typically soluble and "loosely bound" to membranes. There is a slight difference in the kinetic properties of the ghost-bound and the free soluble enzyme. The possible importance of these results for purine uptake and utilization in human red cells is discussed.     

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

Phosphorylation in membranes of intact human erythrocytes.

Studies of phosphorylation in membranes of intact human erythrocytes were performed by incubating erythrocytes in inorganic [32P]phosphate. Analysis of membrane proteins by polyacrylamide gel electrophoresis showed a pattern of phosphorylation similar to that observed when ghost membranes were incubated with [gamma-32P]ATP. Membrane lipid phosphorylation was also similar in intact cells and ghosts. The most heavily phosphorylated lipid, polyphosphoinositide, was closely associated with glycophorin A, the major erythrocyte membrane sialoglycoprotein obtained when the sialoglycoprotein fraction was isolated by the lithium diiodosalicylate-phenol partition procedure. Only 1 molecule of glycophorin A out of every 100 was found to be phosphorylated, and the phosphate exchange occurred specifically in the COOH-terminal intracellular portion of glycophorin A. These studies show that the human erythrocyte can be used as a model for membrane phosphorylation in an intact cell system.     

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.     

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Cytochalasin B does not serve as a marker of glucose transport in rabbit erythrocytes

Glucose inhibitable cytochalasin B binding to erythrocyte membranes has been used as a marker of the glucose transporter. Glucose transport and cytochalasin B binding in rabbit erythrocytes differ from those activities found in human erythrocytes. We evaluated the uptake of 3-0-methylglucose and found similar Km (4.81 +/- 1.20 mM (SEM) and 6.59 +/- 0.72 mM) though significantly different Vmax (5.2 +/- 0.7 nM . min-1/10(9) cells and 234 +/- 13 nM X min -1/10(9) cells, p less than 0.001) for rabbit and human erythrocytes, respectively. Equilibrium binding of cytochalasin B to human erythrocyte membranes demonstrates a high affinity cytochalasin B binding site (Kd 38.6 +/- 22.7 nM) which is displaced by glucose. No comparable glucose inhibitable cytochalasin B site exists for rabbit erythrocyte membranes. Photoaffinity labeling of cytochalasin B confirms the presence of a glucose inhibitable cytochalasin B binding site in human, but not rabbit erythrocyte membranes. Cytochalasin B binding is a useful method in the identification of the glucose transporter in human cells, but the technique may be less useful in other species.     

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.     

Determination of Fluorescence Polarization of Membrane Probes in Intact Erythrocytes: Possible Scattering Artifacts

The anisotropy of the fluorescence of diphenylhexatriene has been reported to be less in the membranes of intact erythrocytes than in erythrocyte ghost membranes or in membranes prepared from erythrocyte lipids. Evidence is presented that this may be an artifact due to the intense light scattering by the intact erythrocytes.     

Localization of the carboxyl terminus of Band 3 to the cytoplasmic side of the erythrocyte membrane using antibodies raised against a synthetic peptide

Polyclonal antibodies were raised in rabbits against a synthetic peptide which corresponds to the 12-amino acid carboxyl-terminal sequence of murine erythrocyte Band 3. Immunoblots of ghost membrane proteins showed that the antibody specifically recognized murine or rat Band 3 but not human or canine Band 3. The antibody also bound to murine ghost membranes applied directly to nitrocellulose but not to human ghost membranes. This shows that the carboxyl terminus of Band 3 is available for antibody binding in ghost membranes and that the carboxyl-terminal sequences of human and mouse Band 3 are not identical. The specificity of the antibody for the carboxyl terminus of Band 3 was confirmed by the loss of antibody binding after digestion of detergent-solubilized ghost membrane proteins with carboxypeptidase Y. In addition, carboxyl-terminal fragments of Band 3 generated by protease treatment of cells or ghost membranes were positive on immunoblots while amino-terminal fragments were negative. In contrast, protease-treated stripped ghost membranes did not contain a carboxyl-terminal fragment of Band 3 that was detectable on immunoblots. The carboxyl terminus of Band 3 was localized to the cytoplasmic side of the erythrocyte membrane since antibody binding as determined by immunofluorescence occurred in ghosts and permeabilized cells but not in intact cells. In addition, competition studies using enzyme-linked immunosorbent assays and immunoblots showed that cells and resealed ghosts competed poorly for antibody compared to ghost membranes, inside-out vesicles, or albumin-conjugated peptide.     

Platelet plasma membrane lectin activity

The lectin activity of human platelet and erythrocyte membranes was evaluated using trypsinized, formalinized erythrocytes from eight species. Platelet membranes had the greatest lectin activity against cow erythrocytes, but also had significant activity against human, sheep, electric eel, and rabbit erythrocytes. In contrast, erythrocyte membranes only had low lectin activity against electric eel erythrocytes with no activity against the other types of erythrocytes tested. The platelet membrane lectin activity was found to reside in protein molecules on the external surface of the platelet plasma membrane. The lectin activity of platelet membranes was inhibited by amino sugars and some basic amino acids: N-acetylated amino sugars and other neutral sugars were without effect. These results demonstrate that the external surface of the platelet plasma membrane has a specific lectin activity.     

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.     

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.     

On the sensitivity of intact cells to perturbation by ethanol

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.