Immunochemical characterization of interactions between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins

Autologous immunoglobulin G present during electrophoresis of human erythrocyte membrane proteins influenced the electrophoretic mobility of some of the proteins. Different types of non-ionic detergents were used for solubilization of the membranes and together with experiments using dimyristoylphosphatidylcholine-derived erythrocyte membrane vesicles this indicated that IgG binds to spectrin, ankyrin, and band 3 protein. The binding was independent on proteolysis and not due to unspecific protein-protein interactions. Immunoblotting experiments also showed binding to polypeptide bands in the spectrin and ankyrin regions and demonstrated the presence of erythrocyte-associated IgG. The reactivity may be due to natural autoantibodies involved in the clearance of cellular debris in vivo. Whether the observations are of relevance for the putative immune-mediated clearance of old erythrocytes from the circulation remains to be established.     

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.     

Erythrocyte membrane elasticity during in vivo ageing

Changes in the ability of senescent erythrocytes to pass through the micro-circulation may cause them to be trapped in the spleen and removed from the blood. To help understand this process we have measured erythrocyte membrane elasticity, to see whether it changes during in vivo ageing. Human and rabbit red cells were fractionated by isopycnic sedimentation to obtain samples of aged and young cells. These were subjected to micropipette analysis in order to determine their membrane shear elastic modulus. We found that the membrane rigidity did not significantly alter as red cells aged. Previously we have also demonstrated that the changed size and shape of aged cells is unlikely to explain their removal from the circulation (Nash, G.B. and Wyard, S.J. (1981) Biorheology, in the press). Thus we conclude that the lifespan of erythrocytes is not determined by factors related to membrane flexibility or cell shape but may depend on changes in their viscous properties (as suggested by Williams, A.R. and Morris, D.R. (1980), Scand. J. Haematol. 24, 57–62).     

Biochemistry of the erythrocyte Rh polypeptides: a review

The clinically important Rh blood group system is complex, consisting of multiple distinct antigens. Despite clinical recognition for over 50 years, the Rh blood group antigens have remained poorly understood on a molecular level until the recent identification and characterization of the "Rh polypeptides," the core structural proteins of the Rh antigens. This group of erythrocyte membrane proteins of molecular weight 30,000-35,000 daltons was first recognized by employing Rh-specific antibodies to immunoprecipitate radiolabeled components of erythrocyte membranes. By using antibodies specific for the Rh D, c, and E antigens, a series of highly related non-identical proteins were immunoprecipitated, indicating that the Rh antigens are composed of multiple related proteins. The Rh polypeptides have been purified and characterized, and they were found to have several unusual biochemical characteristics. The Rh polypeptides penetrate the membrane bilayer; they are linked to the underlying membrane skeleton; they are covalently fatty acid acylated with palmitate. While the Rh antigenic reactivity is unique to human erythrocytes, the Rh polypeptides have been isolated from erythrocytes of diverse species and are thought to be fundamental components of all mammalian erythrocyte membranes. The functional role of the Rh polypeptides remains undefined, but a role in the organization of membrane phospholipid is suspected.     

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.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

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.     

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

Protein and lipid components of the pigeon erythrocyte membrane.

The plasma membrane of the nucleated pigeon erythrocyte was isolated by a method that is simple, reproducible and minimally disruptive, the final preparation consisting of whole cell 'ghosts', recovered at over 40% yield. Alternative methods, which yield membrane fragments, were also tested and some of their possible disadvantages demonstrated. Analysis of the protein components of the isolated membranes by gel elctrophoresis in the presence of sodium dodecyl sulphate revealed that their composition is very similar to that of the proteins of human erythrocyte membranes. However, two major proteins are unique to the nucleated cell membrane; these have apparent mol.wts. of 97000 and 57000. Also, the bands designated 4.2 (74500 mol.wt.) and 6 (35000 mol wt.) by Steck [(1974) J. Cell Biol. 62, 1-19] for the human cell membrane are absent from pigon cell membrane. Glycosylated membrane proteins could not be detected in gels stained with the periodate-Schiff-base procedure. Analysis of membrane phospholipids revealed the same components known to be present in mammalian erythrocytes, though in different proportions. These findings are discussed in the light of known physiological and biochemical differences between avian and mature mammalian erythrocytes.     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Further investigation of methylation on sickle erythrocyte membranes

The methylation of erythrocyte membrane proteins has been investigated with fractionated reversible and irreversible sickle erythrocytes to better understand conflicting results obtained from two laboratories (Green and Kalra (6), Ro et al. (1). When subpopulations of intact erythrocytes obtained by two different separation methods (33% bovine serum albumin and Stractan II gradient centrifugations) were incubated with L-[methyl-3H] methionine at pH 7.2 and 37 degrees C, membranes from both reversible and irreversible sickle erythrocyte populations showed about half the [3H]methyl group incorporation than that observed in normal erythrocytes. In addition, this difference in the level of methylation between normal and sickle cells was maintained during the entire course of a 2-hr incubation utilizing S-adenosyl-L-[methyl-3H]methionine, the immediate in vivo methyl donor.     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

Identification of Surface Proteins on Viable Plasmodium knowlesi Merozoites

Viable merozoites of Plasmodium knowlesi were isolated and the proteins that were labeled on intact merozoites by lactoperoxidase-catalyzed radioiodination were identified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of Triton soluble extracts of labeled merozoites demonstrated eight major bands ranging in apparent molecular weight from 150,000 D to 22,000 D. Exposure of intact merozoites to trypsin (10 μg/ml) for 10 min resulted in the loss of the two highest molecular weight proteins (150,000 D and 105,000 D) and the appearance of two new bands at 70,000 D and 62,000 D. Trypsin treatment under these conditions also removed the receptor(s) for merozoite attachment to erythrocytes. Therefore, these high molecular weight proteins are candidates for the merozoite component that attaches to erythrocytes. There was no evidence that the labeled membrane components were serum or erythrocyte membrane components, two potential contaminants in the preparation. Anti-rhesus erythrocyte antibody did not precipitate labeled merozoite proteins. Furthermore, the immunoprecipitation of labeled merozoite proteins by rhesus anti-merozoite serum was not inhibited by erythrocyte ghosts.     

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.     

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.     

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Isolation and partial characterization of the human erythrocyte band 7 integral membrane protein.

Monoclonal antibodies to the Mr 31,000 major integral membrane protein of the human erythrocyte band 7 region were used to identify the corresponding polypeptide chain and epitope-carrying fragments on immunoblots. Analysis of the erythrocyte membrane, membrane fractions, and cytosol revealed that the Mr 31,000 band 7 integral membrane protein is unique and not related to any of the other water-soluble or membrane-bound band 7 components. Cross-reacting proteins were identified in the membranes of other mammalian erythrocytes and in cell lines of epithelial and lymphoid origin. Proteolytic digestion of intact human erythrocytes or erythrocyte membranes demonstrated that the band 7 integral membrane protein has an intracellular domain larger than Mr 12,000; it does not have an extracellular one. One of the monoclonal antibodies was employed for the isolation of band 7 integral membrane protein by immunoaffinity chromatography; subsequent Edman degradation revealed a blocked N-terminus.     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.