The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above.We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Resolution of the paradox of red cell shape changes in low and high pH

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.     

Fluidity of erythrocyte membranes from patients with Duchenne muscular dystrophy: studies in intact erythrocytes and in ghosts

We have studied the alterations of fluidity in intact erythrocytes and in erythrocyte membranes from patients with Duchenne muscular dystrophy. The interest of this study was to comparison directly two types of results; these demonstrate an increase of fluidity in the erythrocytic membranes, no changes are present when the label is incorporated in intact erythrocytes. It might be inferred that hypotonic haemolysis removes components that are more weakly bound in Duchenne membranes, and that exert an immobilizing effect on the membrane lipids.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. I. General properties of variously prepared membranes and the mechanism of the isosmotic imidazole effect.

1. Membranes prepared from human erythrocytes hemolyzed in isosmotic (310 imosM) imidazole buffer, pH 7.4, show enhanced and stabilized (Ca2+ + Mg2+)-ATPase activity compared with membranes prepared from erythrocytes hemolyzed in hypotonic (20 imosM) phosphate or imidazole buffer, pH 7.4. 2. Exposure of intact erythrocytes or well-washed erythrocyte membranes to isosmotic imidazole does not cause enhanced (Ca2+ + Mg2+)-ATPase activity. 3. Exposure of erythrocyte membranes, in the presence of isosmotic imidazole, to the supernatant of erythrocyte hemolysis or to a partially purified endogenous (Ca2+ + Mg2+)-ATPase activator, promotes enhanced (Ca2+ + Mg2+)-ATPase activity. Under appropriate conditions, NaCl can be shown to substitute for imidazole. The results demonstrate that imidazole does not act directly on the erythrocyte membrane but rather by promoting interaction between an endogenous (Ca2+ + Mg2+)-ATPase activator and the erythrocyte membrane.     

Changes in membrane fluidity of erythrocytes during cell maturation

The measurements of the fluorescence polarization of perylene embedded in erythrocyte membranes were carried out with normal and reticulocyte-rich blood, and the microviscosity of erythrocyte membranes was calculated from the polarization degree. In intact cells, reticulocyte membranes had a significantly lower microviscosity than normal erythrocyte membranes, while in ghosts no significant difference in membrane microviscosity was observed between reticulocytes and mature erythrocytes.     

Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.     

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.     

Sendai Virus Fusion Activity as Modulated by Target Membrane Components

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.     

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.     

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

Stomatocytosis of latex particles (0.26 micron) by rat erythrocytes by the electrical breakdown technique

The uptake of macromolecules by erythrocytes can be achieved with the electrical breakdown technique [2, 4]. In this technique the erythrocyte membranes are subjected to a high external electrical field pulse for a short period. Local, reversible breakdowns of the cell membrane occur above a critical field strength which lead to a time-dependent increase in the permeability of the membrane. By this means, human erythrocyte membranes can be made permeable to DNA, pharmaceutical compounds, and latex particles following an electrical field pulse [1, 3, 5]. Larger particles should also be taken up by erythrocytes using this method. Vienken et al. [5] demonstrated the entrapment of latex particles with a diameter of 0.091 micron in human erythrocyte ghosts, although this was shown with only a single electron micrograph which does not prove that the ghost membrane was intact. In our experiments in order to entrap latex particles with a diameter of 0.26 micron rat erythrocytes were subjected to an electrical field pulse of 12 kV/cm with a decay time of 60 microseconds. Experiments using the electron microscope show that after such an electrical field pulse the uptake of latex particles by rat erythrocytes follows the stomatocytotic pathway. We show further that using electron microscopic techniques, a single section cannot demonstrate the completed uptake of a latex particle by the erythrocyte.     

Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.

Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.     

Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats

In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116, 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4 degrees C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat-erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF-receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients.     

Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte plasma membrane.     

Structural effects of titanium citrate on the human erythrocyte membrane

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.     

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.