Glucose 6-phosphate dehydrogenase activity in membranes of erythrocytes from normal individuals and subjects with Mediterranean G6PD deficiency

Unsealed, hemoglobin-free erythrocyte ghosts contain low yet significant levels of Glucose 6-phosphate dehydrogenase (G6PD) activity. This activity is comparable in erythrocyte ghosts obtained from normal individuals and from G6PD-deficient subjects (of Mediterranean type), in spite of the marked differences found in the corresponding cytosolic compartments. The membrane preparations can bind purified human G6PD (type B) to their cytoplasmic surface according to patterns of positive cooperativity. 2.4 × 10⁴ and 1.6 × 10⁴ G6PD-binding sites are present on the inner surface of each ghost obtained from normal and from G6PD-deficient erythrocytes, respectively, the relevant association constants being 2.8 × 10⁶ M^?1 and 0.82 × 10⁶ M^?1. The interaction of G6PD with the ghosts is unaffected by different ionic strengths or by metabolites such as glucose 6-phosphate, NADP and NADPH.     

An acid protease in human erythrocytes and its localization in the inner membrane.

The isolation of erythrocytes of high purity from human blood was achieved by a combination of the two well established methods cells in erythrocyte preparations of different purities was studied. The acid protease activity was recovered to a level comparable with the recovery of erythrocytes, while the neutral protease activity as detected by the release of acid-soluble peptides from hemoglobin or casein disappeared in proportion to the removal of white blood cells. An acid protease was solubilized from the membranes of the purified erythrocytes by the extraction with 1-butanol. The enzyme was active in a pH range from 2 to 4, and sensitive to pepstatin. It was named pH-3 protease after its pH optimum. Sealed ghosts with right-side-out membranes and inside-out vesicles with reverted membranes were prepared from the purified erythrocytes and compared with respect to pH-3 protease activity for its latency as well as its inactivation by tryptic digestion. The results obtained indicate that pH-3 protase is localized on the inner surface of erythrocyte membranes. The self-digestion experiments at pH 4 using the sealed ghosts showed higher availability to pH-3 protease of spectrin and IVa protein than the other membrane proteins, also suggesting the localization of an acid protease in the inner membranes of erythrocytes.     

Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.     

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.     

Membrane orientation of sheep spectrin

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membrane localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected immunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.     

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.     

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

Plasmodium falciparum: analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane

The location of the Plasmodium falciparum vaccine candidate antigen Pf155/RESA in the membrane of infected erythrocytes was analzyed by means of selective surface radioiodination and immunofluorescence of surface-modified cells. The lack of radiolabel in Pf155/RESA as well as its localization by immunofluorescence similar to that of the N-terminal region of erythrocyte band 3 suggests that the antigen is associated with the cytoplasmic phase of the erythrocyte membrane. In concordance with this, Pf155/RESA was detected by immunofluorescence on the surface of inside out membrane vesicles from P. falciparum-infected erythrocytes. Pf155/RESA from spent culture medium also bound to inside out membrane vesicles of normal erythrocytes as well as to cytoskeletal shells of such vesicles, but failed to bind to sealed right-side out membrane vesicles. Depletion of spectrin from the vesicles abolished antigen binding, suggesting that Pf155/RESA association with the erythrocyte cytoskeleton is mediated by spectrin.     

Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.     

Comparative distribution of glyceraldehyde 3-phosphate dehydrogenase activity in human, guinea-pig, rabbit and mouse erythrocytes

1. Guinea-pig erythrocytes contain half the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) present in human cells. About 60% of their total activity is membrane-bound. 2. Rabbit erythrocytes also contain half the amount of the enzyme of human red cells. The distribution of G3PD in rabbit cells, however, is similar to that of human cells with 70% of the enzyme being membrane-bound. 3. Mouse erythrocytes contain about two-thirds of G3PD activity present in human cells. All their enzyme activity is present in membrane-free hemolysate. 4. Non-human erythrocyte membrane proteins, in addition, have relatively greater amount of band 2.1, lack band 2.2, have a more heterogenous band 3 than its human counterpart, and have overlapping bands 4.1 and 4.2.     

Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Membrane phospholipid asymmetry as a factor in erythrocyte-endothelial cell interactions

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.     

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.     

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Summary Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37°C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37° for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.     

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.     

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 size of erythrocyte ghosts

The volume of resealed erythrocyte ghosts formed during hypotonic hemolysis of normal human erythrocytes was measured by means of a continuous mean corpuscular volume analyzer. The final volume of resealed ghosts was 140.6 ± 15.2 fl. Strong correlations exist between the volume of ghosts and the initial mean corpuscular volume and mean corpuscular hemoglobin of the erythrocyte, and between the enlargement ratio and the mean corpuscular volume or mean corpuscular hemoglobin of the erythrocyte.     

Water exchange through erythrocyte membranes: p-choloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes . The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37°C. No inhibition was noticed after short incubation at 0°C as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance ( n . m . r . ) method appears as a useful tool for studying changes in water diffusiofl in erythrocyte ghosts with the aim of locating the water channel.     

Rotational diffusion of band 3 in erythrocyte membranes. 2. Binding of cytoplasmic enzymes

Time-resolved phosphorescence anisotropy has been used to study the rotational diffusion of eosin-labeled human erythrocyte band 3 in the presence of an enzyme bound at its cytoplasmic pole. With increasing amounts of G3PD (glyceraldehyde-3-phosphate dehydrogenase) added to ghosts, the infinite time anisotropy (r infinity) increases, and at saturating concentrations, very little decay of the anisotropy r(t) occurs at all. These phenomena are reversed by elution of the enzyme with 150 mM NaCl. Prior proteolytic removal of the N-terminal 41-kDa cytoplasmic fragment of band 3 also disenables the G3PD effect. When ghosts are stripped of their residually bound G3PD, a small reduction in the fraction of immobile band 3 is observed. Finally, titration of band 3 sites with aldolase shows effects on the r(t) qualitatively similar to those observed with G3PD. On the basis of our interpretation of the heterogenous anisotropy decay of eosin-labeled band 3 [Matayoshi, E. D., & Jovin, T. M. (1991) Biochemistry (preceding paper in this issue)], we conclude that the binding of G3PD and aldolase results in the immobilization of band 3 oligomers.     

Antibodies against Citrobacter braakii O37 cells recognize the N-glycan of the band 3 glycoprotein of human erythrocyte membrane

The phenomenon of molecular mimicry was found previously for Citrobacter braakii O37, which shared epitopes with human and horse erythrocytes. The aim of this study was to elucidate the basis of the serological cross-reactivity between anti-C. braakii O37 serum and human erythrocytes. The experiments involved analyzing the epitope on the human erythrocyte membrane, that could be recognized by affinity-purified antibodies. The results indicated a specific glycoprotein fraction in immunoblotting, namely band 3, which interacted with the antibodies purified on lipopolysaccharide from C. braakii O37 LPS (LPS O37) and its core affinity columns. Treating the erythrocytes with trypsin, which cleaves glycophorin A, improved the agglutination because band 3 became more available for antibody binding. Isolated band 3 immobilized on an affinity plate could be used to purify antibodies from the anti-C. braakii O37 serum. These antibodies showed a specific reactivity to LPS O37, but not to the related lipopolysaccharide from Salmonella Toucra O48. Furthermore, the inhibition of agglutination with lactose, the diminished interaction of the specific antibodies purified on LPS O37 with endo-beta-galactosidase-treated band 3, and the reactivity of these antibodies to the 40-kDa fragment of band 3 but not to its trypsin-elaborated 60-kDa fragment, all indicated that the epitope is located on the N-glycan of band 3.