Morphology of haemoglobin-containing human erythrocyte ‘ghosts’

Resealed erythrocyte ‘ghosts’ have been proposed as in vivo carriers for enzymes in the therapy of inherited metabolic diseases. A long life-span of this carrier is required when the erythrocyte ‘ghost’ is intended to be the site of substrate degradation in the circulation. Erythrocyte ‘ghosts’ have been prepared that show cell content and membrane transport characteristics that are closely similar to those of normal erythrocytes. Since the morphology of these ‘ghosts’ could probably also affect the in vivo life-span, haemoglobin-containing human erythrocyte ‘ghosts’ have been studied using scanning electron-microscopy.Preparation of the erythrocyte ‘ghosts’ involved a method of hypo-osmotic dialysis and consecutive iso-osmotic dialysis. Samples for scanning electron microscopy were prepared by fixation in glutaraldehyde, with post-fixation in osmium tetroxide. Dehydration was obtained by increasing concentrations of ethanol and critical-point-drying. Control experiments with normal erythrocytes showed no major artefacts by the methods used. Erythrocyte ‘ghosts’ showed polymorphic shapes. Two thirds were stomatocytes, but echinocytes and cells with deep invaginations or inter-twisted infoldings were also observed. Distribution of the different cell types could be affected by the preparation techniques used. Washing of the erythrocyte ‘ghosts’ at very low centrifugation speeds resulted in 60% of the cells appearing as biconcave discoids and one third as stomatocytes.The results demonstrate that the preparation disturbed the balance of the biconcave shape of resealed erythrocyte ‘ghosts’. Minor alterations of the methods allowed the preparation of erythrocyte ‘ghosts’, the majority of which showed morphology closely similar to that of normal erythrocytes. Attention must be given to all preparation factors to avoid irreversible damage and therefore prior to use of these cells in patients or when methods are altered, the morphology of these cellular carriers must be closely controlled.     

AS1411 aptamer tagged PLGA-lecithin-PEG nanoparticles for tumor cell targeting and drug delivery

Liposomes and polymers are widely used drug carriers for controlled release since they offer many advantages like increased treatment effectiveness, reduced toxicity and are of biodegradable nature. In this work, anticancer drug‐loaded PLGA‐lecithin‐PEG nanoparticles (NPs) were synthesized and were functionalized with AS1411 anti‐nucleolin aptamers for site‐specific targeting against tumor cells which over expresses nucleolin receptors. The particles were characterized by transmission electron microscope (TEM) and X‐ray photoelectron spectroscopy (XPS). The drug‐loading efficiency, encapsulation efficiency and in vitro drug release studies were conducted using UV spectroscopy. Cytotoxicity studies were carried out in two different cancer cell lines, MCF‐7 and GI‐1 cells and two different normal cells, L929 cells and HMEC cells. Confocal microscopy and flowcytometry confirmed the cellular uptake of particles and targeted drug delivery. The morphology analysis of the NPs proved that the particles were smooth and spherical in shape with a size ranging from 60 to 110 nm. Drug‐loading studies indicated that under the same drug loading, the aptamer‐targeted NPs show enhanced cancer killing effect compared to the corresponding non‐targeted NPs. In addition, the PLGA‐lecithin‐PEG NPs exhibited high encapsulation efficiency and superior sustained drug release than the drug loaded in plain PLGA NPs. The results confirmed that AS1411 aptamer‐PLGA‐lecithin‐PEG NPs are potential carrier candidates for differential targeted drug delivery. Biotechnol. Bioeng. 2012; 109: 2920–2931. © 2012 Wiley Periodicals, Inc.     

Proteolysis of N-ethylmaleimide-modified aldolase loaded into erythrocyte ghosts: prevention by inhibitors of calpain.

1. When rabbit muscle aldolase labelled with tritium and inactivated by N-ethylmaleimide (NEM) was loaded into erythrocyte ghosts, significant proteolysis of the loaded protein occurred. The major product of this proteolysis, separated by electrophoresis under dissociating conditions, was found to be approx. 2 kDa smaller than the parent protein. 2. Proteolysis was detectable during erythrocyte ghost loading at 0 degrees C, reaching a plateau after approx. 12 min. Subsequent incubation at 37 degrees C to allow resealing of the ghosts resulted in additional proteolysis, and up to 20% of the loaded protein was converted to the smaller 38 kDa derivative. 3. EDTA, EGTA, leupeptin and chymostatin, each inhibitors of calcium-activated neutral proteinases (calpains), were the most effective inhibitors of the proteolysis of NEM-inactivated aldolase in ghosts. Other proteinase inhibitors were ineffective, while phenylmethanesulphonyl fluoride was only partially effective. 4. Inhibition of the proteolysis by EGTA was prevented by CaCl2, supporting the involvement of erythrocyte calpain. 5. Pretreatment of ghosts with EGTA prior to loading of NEM-modified aldolase followed by microinjection of the protein into HeLa cells did not result in a different rate of its overall breakdown to acid-soluble products. EGTA is suggested as a useful agent for the erythrocyte ghost-mediated microinjection of calpain-sensitive proteins.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Triacylglycerol lipase in bovine erythrocytes

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.     

Insulin causes insulin-receptor internalization in human erythrocyte ghosts.

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.     

Complement-mediated lysis of pigeon erythrocyte ghosts analysed by flow cytometry. Evidence for the involvement of a ''threshold'' phenomenon.

Flow-cytometric analysis of complement-mediated lysis of antibody-coated pigeon erythrocyte ghosts containing fluorescein was carried out to determine whether lysis involved a gradual release of fluorescein or a 'threshold' release from individual cells. Antibody-coated ghosts were comprised of three subpopulations identified by fluorescence and scatter (size). These were: (a) highly fluorescent, medium scatter, (b) medium fluorescence, high scatter, and (c) low (or zero) fluorescence, low scatter. Lysed ghosts and isolated nuclei were identified by fluorescence microscopy and scanning electron microscopy. Fluorescence distributions analysed by flow cytometry indicated that, after complement attack, those ghosts remaining intact retained all their fluorescent label. A time course of changes in ratios of the three subpopulations indicated that once lysis of an individual ghost was initiated, release of label was complete within 1 min; no stages of intermediary fluorescence appeared, and those ghosts remaining at the end of the experiment retained the same fluorescence intensity as control ghosts. The results supported the hypothesis that complement-mediated cell lysis is a 'threshold' phenomenon; a submaximal response by a cell population representing a complete response by only some of the cells rather than a partial response by all of the cells.     

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.     

Altering erythrocyte membrane composition with phospholipid exchange protein

• 1.1. In establishing a protocol for the use of a phospholipid (PL) 2 exchange protein (PLEP) preparation to incorporate fluorescent lipids into membrane fragments, the potential for the PLEP preparation to alter membrane composition was investigated.
• 2.2. Cholesterol was removed from erythrocyte ghosts and PL content was increased after incubation of the membranes with phosphatidylcholine (PC) vesicles and a nonspecific bovine liver PLEP preparation.
• 3.3. The elevated PL/cholesterol ratio of the ghosts decreased fluorescence polarization of diphenylhexatriene (DPH), indicating an increased average membrane fluidity.
• 4.4. The increased PL/cholesterol ratio and reduced DPH fluorescence polarization were prevented by including cholesterol in the PC vesicles during the initial incubation of ghosts with PLEP preparation and lipid.
• 5.5. In addition, the PC content of ghost membranes was elevated after incubation with PLEP preparation and either PC or mixed PC-cholesterol vesicles.

     

Excretion of superoxide by phagocytes measured with cytochrome c entrapped in resealed erythrocyte ghosts

Resealed erythrocyte membranes (ghosts) filled with (Fe3+)cytochrome c were used as an assay system to measure the release of superoxide (O-2) from human phagocytes into the incubation medium. Neutrophils, activated by either opsonized zymosan particles or the soluble stimulus phorbol myristate acetate, released O-2, which subsequently entered the ghosts and reduced (Fe3+)cytochrome c. This reaction was dependent on the time of incubation, the concentration of neutrophils, the concentration of stimulus, and the concentration of ghosts. The reaction was completely inhibited by superoxide dismutase and by 4,4'-diisothiocyano-2,2'-disulfonic acid, a specific blocker of anion channels in membranes. The reduction of (Fe3+)cytochrome c free in solution was about four times as fast as the reduction of (Fe3+)cytochrome c in the ghosts. Human eosinophils stimulated by phorbol myristate acetate reacted similarly to human neutrophils; the rate of O-2 production/cell was about twice as high for eosinophils as for neutrophils. In contrast, eosinophils stimulated with opsonized zymosan particles only reduced (Fe3+)cytochrome c free in solution, but not (Fe3+)cytochrome c in ghosts. This lack of reaction was not due to production of an inhibitor or below threshold generation of O-2 for the ghost assay. These results indicate: 1) activated human neutrophils and eosinophils can release O-2 or a similar product into the incubation medium; and 2) reduction of (Fe3+)cytochrome c free in solution is no proof for O-2 excretion by phagocytes.     

Albumin's Influence on Carprofen Enantiomers–Hymecromone Interaction

Hymecromone is an important coumarin drug, and carprofen is one of the most important nonsteroidal antiinflammatory drugs (NSAIDs). The present study aims to determine the influence of bovine serum albumin (BSA) on the carprofen–hymecromone interaction. The inhibition of carprofen enantiomers on the UDP‐glucuronosyltransferase (UGT) 2B7‐catalyzed glucuronidation of hymecromone was investigated in the UGTs incubation system with and without BSA. The inhibition capability of increased by 20% (P < 0.001) of (R)‐carprofen after the addition of 0.5% BSA in the incubation mixture. In contrast, no significant difference was observed for the inhibition of (S)‐carprofen on UGT2B7 activity in the absence or presence of 0.5% BSA in the incubation system. The Lineweaver‐Burk plot showed that the intersection point was located in the vertical axis, indicating the competitive inhibition of (R)‐carprofen on UGT2B7 in the incubation system with BSA, which is consistent with the inhibition kinetic type of (R)‐carprofen on UGT2B7 in the incubation system without BSA. Furthermore, the second plot using the slopes from the Lineweaver‐Burk versus the concentrations of (R)‐carprofen showed that the fitting equation was y=39.997x+50. Using this equation, the inhibition kinetic parameter was calculated to be 1.3 μM. For (S)‐carprofen, the intersection point was located in the horizontal axis in the Lineweaver‐Burk plot for the incubation system with BSA, indicating the noncompetitive inhibition of (S)‐carprofen on the activity of UGT2B7. The fitting plot of the second plot was y=24.6x+180, and the inhibition kinetic parameter was 7.3 μM. In conclusion, the present study gives a short summary of BSA's influence on the carprofen enantiomers–hymecromone interaction, which will guide the clinical application of carprofen and hymecromone. Chirality 28:226–229, 2016. © 2015 Wiley Periodicals, Inc.     

Separation and determination of dexamethasone sodium phosphate in cochlear perilymph fluid by liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization

The method for separation and determination of dexamethasone sodium phosphate (DexP) in cochlear perilymph fluid (CPF) of cavy was developed using HPLC with ultraviolet (UV) monitoring and electrospray ionization/mass spectrometry (ESI/MS) identification. The quantitative determination of DexP in CPF was achieved by HPLC with UV detection at 245 nm. The separation was carried out on a Phenomenex ODS(3) column ( 250 mm x 4.6 mm i.d., 5 microm) with the mobile phase of acetonitrile-5mmol/l ammonium acetate (23:77 (v/v)) at a flow rate of 1.0 ml/min. DexP was baseline separated from the matrices of CPF blanks within 15 min. The linearity ranged from 0.5 to 50 microg/ml. The limit of detection was 0.10 microg/ml. The recovery ranged from 98.5 to 100.8%. The relative standard deviations (R.S.D.s) of intra- and inter-day peak area were between 0.7-1.3 and 1.2-3.5%, respectively. Both full scan MS and MS2 of DexP with positive and negative polarity were obtained and elucidated. The specific ions were chosen to characterize DexP in the CPF sample. Using the proposed HPLC-UV-ESI/MS method, the concentration of DexP in CPF samples after both vein and middle ear injections were determined, and the relationships between concentration and time were obtained. This method offered reference data for clinical investigation of DexP to cure ear diseases.     

Inhibition of complement-induced [14C]sucrose release by intracellular and extracellular monoclonal antibodies to C9: Evidence that C9 is a transmembrane protein

Monoclonal antibodies to the terminal component of the human complement pathway, C9 were used to inhibit the complement-induced release of entrapped [¹⁴C]sucrose from erythrocyte ghosts. Antibodies were present either outside, or entrapped within the ghosts. Different monoclonal antibodies were demonstrated to inhibit [¹⁴C]sucrose release depending on whether the antibody was outside or entrapped within the ghosts. These findings demonstrate that C9 within the membrane attack complex on erythrocyte membranes is an asymmetrical transmembrane protein penetrating into the cytoplasmic space.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

A comparison between porcine,ovine, and bovine intervertebral disc anatomy and single lamella annulus fibrosus tensile properties

This project aimed to compare gross anatomical measures and biomechanical properties of single lamellae from the annulus fibrosus of ovine and porcine lumbar vertebrae, and bovine tail vertebrae. The morphology of the vertebrae of these species differ significantly both from each other and from human, yet how these differences alter biomechanical properties is unknown. Geometric parameters measured in this study included: 1) absolute and relative intervertebral (IVD) and vertebral body height and 2) absolute and relative intervertebral disc (IVD) anterior‐posterior (AP) and medial‐lateral (ML) widths. Single lamella tensile properties included toe‐region stress and stretch ratio, stiffness, and tensile strength. As expected, the bovine tail IVD revealed a more circular shape compared with both the ovine and porcine lumbar IVD. The bovine tail also had the largest IVD to vertebral body height ratio (due to having the highest absolute IVD height). Bovine tail lamellae were also found to be strongest and stiffest (in tension) while ovine lumbar lamellae were weakest and most compliant. Histological analysis revealed the greatest proportion of collagen in the bovine corroborating findings of increased strength and stiffness. The observed differences in anatomical shape, connective tissue composition, and tensile properties need to be considered when choosing an appropriate model for IVD research. J. Morphol. 277:244–251, 2016. © 2015 Wiley Periodicals, Inc.     

Membrane perturbations of erythrocyte ghosts by spectrin release

The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.     

Dopamine‐loaded poly(d,l‐lactic‐co‐glycolic acid) microspheres: New strategy for encapsulating small hydrophilic drugs with high efficiency

The effective controlled release of small hydrophilic drugs from poly(d ,l ‐lactic‐co‐glycolic acid) (PLGA) microspheres has remained a challenge, largely due to the difficulty of loading a large amount of the drug inside the microspheres, owing to the hydrophilicity of the drugs. This study provides a new strategy for increasing encapsulation of small hydrophilic drugs inside PLGA microspheres by utilizing noncovalent, physical adsorption between hydrophilic drugs and emulsifying polymers of poly(vinyl alcohol) and pluronic. An order of magnitude increase in drug loading efficiency from 2.7 to 18.6% for dopamine, a model small hydrophilic drug, was achieved. The large amount of dopamine‐loaded PLGA formulation herein could be useful for the treatment of Parkinson's disease. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:215–223, 2014     

5-Fluorouracil-Loaded BSA Nanoparticles: Formulation Optimization and In Vitro Release Study

Over the past few decades, there has been considerable interest in developing protein nanoparticles as drug delivery devices. The underlying rationale is their exceptional characteristics, namely biodegradability and nonantigenicity. Herein, phase separation method was used to prepare 5-fluorouracil-loaded bovine serum albumin (BSA) nanoparticles. Drug release was tracked by continuous flow dialysis technique. Effect of process variables on loading efficiency of 5-fluorouracil was investigated and optimized through Taguchi’s M16 design with the amount of entrapped drug as response. Optimum condition was found to be 2 mg/mL of 5-fluorouracil, 3.7 mL of added ethanol, 176 μL of glutaraldehyde, drug–protein incubation time of 30 min, and pH of 8.4 for 200 mg of BSA in 2 mL drug solution. pH had the most noticeable effect on the amount of entrapped drug, but glutaraldehyde had the least. Mean diameter and zeta potential of fabricated nanoparticles under these conditions were 210 nm and −31.7 mV, respectively. Drug-loaded BSA nanoparticles suspension maintained constant release of drug for 20 h under experimental conditions, so this colloidal drug carrier is capable of releasing drug in a sustained manner.     

Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine [ epsilon-N(L-furoylmethyl)-L-lysine]

Non-enzymatic glycosylation of erythrocyte membranes was studied using a non-radioactive and sensitive procedure for specific quantitation of lysine-bound glucose in proteins. About 2 nmol lysine-bound glucose/mg protein were found in ghosts from normal erythrocytes, and this value was about doubled in diabetic patients. In vitro incubation of normal ghosts with glucose gave rise to levels of lysine-bound glucose similar to those found in diabetics. There was a linear correlation between the amount of lysine-bound glucose of total hemoglobin and of membrane proteins. Membrane glycosylation also depended on the age of erythrocytes displaying significantly higher values in old cell populations.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.