Surface Of Erythrocyte Membrane Research Articles

Spin-label detection of hemoglobin-membrane interaction at physiological pH.

The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a significant hemoglobin-membrane interaction which exhibits a substantial hemoglobin concentration dependence over the concentration range 0-12 mg/mL. An equilibrium binding model yields a hemoglobin-membrane dissociation constant, Kd, on the order of 10(-4) M, at and above physiological pH; the interaction is classified as very low-affinity binding. The interaction increases significantly when the pH is decreased. Half-saturation of the binding sites occurs at a ratio of about 10(8) hemoglobins per cell.

Cross-linking of hemoglobin to the cytoplasmic surface of human erythrocyte membranes. Identification of band 3 as a site for hemoglobin binding in Cu2+-o-phenanthroline catalyzed cross-linking.

The addition of hemoglobin to isolated membrane ghosts of human erythrocytes followed by catalytic oxidation with Cu2+-o-phenanthroline results in the covalent attachment of hemoglobin to the membrane. A decrease in the mobility of Band 3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to an increase in molecular weight of approximately 16,000 suggests that Band 3 is a site for the covalent attachment of hemoglobin to the membrane under these conditions. This conclusion was confirmed using two-dimensional gels in which the covalent linkage was cleaved with 2-mercaptoethanol in the second dimension. 14C-labeled hemoglobin was used to show that the new band was composed of one globin monomer per Band 3 monomer. Additionally, it was demonstrated that only the beta chain of hemoglobin is covalently bound to the Band 3 protein.

The interaction of encephalomyocarditis virus with its erythrocyte receptor on affinity chromatography columns.

Glycophorin, the major sialoglycoprotein in the human erythrocyte surface membrane, can serve as a red cell receptor for both wheat-germ agglutinin (WGA) and encephalomyocarditis (EMC) virus since glycophorin bound to WGA--Sepharose can at the same time bind EMC virus. In contrast, glycophorin bound to WGA--Sepharose cannot bind EMC virus in the presence of SDS. The evidence suggests that virus binding to glycophorin-WGA--Sepharose occurred in the absence of SDS because glycophorin was present in aggregated complexes which were large enough either to accommodate both EMC virus and WGA at the same time, or alternatively to provide sufficient attachment sites for multivalent binding of virions.

Erythrocyte membrane sidedness in lectin control of the Ca2+-A23187-mediated diskocyte goes to and comes from echinocyte conversion.

Increasing the cytoplasmic calcium concentration of human erythrocytes with ionophore A23187 drives the transformation from diskocyte to echinocyte morphology1. This transformation is closely linked to the intracellular ATP level, which drops when calcium is introduced across the membrane2,3. The echinocyte morphology reverts to the diskocyte after restoration of normal ATP and calcium levels4,5. ATP control of cell morphology is thought to depend on the action of a protein kinase and phosphatase which reversibly modify the membrane cytoskeleton6–9. The cytoskeleton in turn is in contact with the outside membrane surface via transmembrane proteins10–11. We show here that binding of the lectin, wheat-germ agglutinin (WGA), to the erythrocyte membrane surface blocks the morphological conversions diskocyte ⇄ echinocyte in both directions. WGA seems to work via a mechanism involving the transmembrane glycoprotein, glycophorin13,14.

A spin label study of erythrocyte membranes during simulation of freezing.

Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.

Interaction of Sendai virus proteins with the cytoplasmic surface of erythrocyte membranes following viral envelope fusion.

Radiolabeled Sendai virus was allowed to fuse with human erythrocytes and inside out vesicles were prepared from the erythrocyte membranes. Viral proteins incorporated into inside out vesicles were released from the membrane following treatment with various agents which perturb protein structure. Disruption products were analyzed by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the viral proteins. The results indicated that 6 M guanidine, 40 mM lithium diiodosalicylate (pH 11-13), and 4 M potassium thiocyanate removed viral nucleocapsid and M proteins from the vesicles. Urea (6.0 M) and 5 mM p-chloromercuribenzenesulfonate removed only viral nucleocapsid proteins. KCl (1.0 or 0.1 M), 5 or 0.5 mM sodium phosphate, 10 mM EDTA (pH 10), 10 mM lithium diiodosalicylate, 1 M urea, 0.5 mM ATP, or freeze-thaw cycles did not release any viral proteins. By contrast, treatment of inside out vesicles with Triton X-100 solubilizes the lipid bilayer, releasing viral integral membrane glycoproteins and leaving viral nucleocapsids intact. Nucleocapsids isolated from virions do not adsorb nonspecifically to control inside out vesicles. These data imply that Sendai viral nucleocapsid and M proteins interact extensively with the cytoplasmic surface of infected cell membranes, in the manner of peripheral membrane proteins, whereas the glycoproteins, HN and F1, behave as integral membrane proteins.

In vivo effects of PUVA on lymphocytes function.

Whole-body exposure to erythemogenic doses of PUVA in normal volunteer subjects resulted in a decrease in the proportion of circulating sheep erythrocyte rosette-forming and surface membrane immunoglobulin-positive lymphocytes and an increase in the proportion of null cells. Doses of PUVA resulting in minimal or no erythema had similar but less marked effects on lymphocytes. The changes appeared 30 min after exposure, were maximal at 12-16 h and returned to pre-treatment levels by 72 h post-irradiation. The response of lymphocytes to stimulation with phytohaemagglutinin was unaffected. No alteration occurred in the absolute numbers of circulating lymphocytes or polymorphonuclear leukocytes.

Acute lymphocytic leukemia and complement receptors. (A study of 45 cases).

The expression of complement receptors (C3R) associated or not to sheep erythrocyte receptors (ER) and surface membrane immunoglobulins (SmIg) was determined on lymphoblasts of 45 patients with acute lymphocyte leukemia (ALL) at onset and/or in the first relapse. We found C3R simultaneously expressed with ER or SmIg on lymphoblasts of T cell ALL and B cell ALL. Lymphoblasts were positive for C3R in absence of ER and SmIg only in three patients in the hematological relapse. We stress the importance to find C3R as independent marker on ALL cells at onset and in subsequent relapses.

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus.

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double-labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.

Binding of lipoteichoic acid of group A streptococci to isolated human erythrocyte membranes

The spontaneous binding of group A streptococcal lipoteichoic acid (LTA) to mammalian cell membranes was studied in isolated membranes of human erythrocytes. The binding of radiolabeled LTA to erythrocyte membranes was dependent on membrane concentration and time. Binding approached a maximum within 30 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. The displaced LTA was eluted from a column of Sepharose 6B in a position identical to that of authentic LTA, suggesting that binding did not alter the size of the molecule. A dissociation constant of 42 micrometers was calculated, and only one population of approximately 5.5 X 10(6) binding sites per erhtyrocyte membrane was detected. Since these results suggested that erythrocyte membranes possess specific binding sites for LTA, an attempt was made to localize the putative receptors to the outside or the inside surface of the erhtyrocyte membrane. Assays of the binding of LTA to resealed right-side-out and inside-out membrane ghosts demonstrated that the outside surface was able to bind over 10 times more LTA than the inside surface. These results support the concept that the membranes possess specific binding sites for LTA and inciate that these binding sites are located almost entirely on the outside surface of erythrocyte membranes.

A regulatory component of adenylate cyclase is located on the inner surface of human erythrocyte membranes

Summary Human erythrocyte membranes contain the guanine nucleotide regulatory component of adenylate cyclase termed N, which can be assayed by in vitro complementation of adenylate cyclase in membranes of the cyc − variant of the S49 lymphoma cell. We used inside-out (IO) and rightside-out (RO) membrane vesicles to determine the membrane orientation of N: Trypsin inactivates N in IO but not in RO vesicles. Treatment with cholera toxin and NAD + activates N in IO but not in RO vesicles. These experiments provide strong evidence that N is located on the inner surface of the human erythrocyte membrane.

Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.

Surface origin of erythrocytic chalone in vitro in experimental polycythemia

Tests on surviving cultures of bone marrow cells from rats with experimental polycythemia showed that the chalone activity of, erythrocytic chalone is considerably reduced in the presence of phytohemagglutinin (PHA). The chalone depresses the agglutinating activity of PHA on bone marrow cells. Adsorption of the chalone preparation on immobilized PHA leads to disappearance of electrophoretically recorded PAS-positive bands from it and a sharp decrease in the intensity of the PAS-negative band. Experiments with preliminary incubation on rat erythrocytes before isolation of the chalone preparation suggest that in the course of its isolation two polypeptides are selectively liberated into the medium, one of them being PAS-positive. It is suggested that erythrocyte surface membrane proteins, are components of the chalone. The possible mechanism of liberation of chalones from the surface of the cells and the particular features of their action on cells are discussed.

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.

Diazotized [ 125I]diiodosulfanilic acid as a label for cell surface membranes. Studies on erythrocytes

1. 1. Diazotized 2,6-diiodosulfanilic acid (DDISA) appears to have properties suitable to serve as an artificial, non-penetrating label of cell surface membranes. Therefore, the conditions for selective labeling of cell surface membranes as compared to intracellular proteins as well as a method for its chemical determination were explored in the present study. 2. 2. DDISA reacts with α-naphthol at neutral pH to produce a compound (1-hydroxy-4-(2,6-diiodo-4-sulfo-1-phenylazo-(naphthylene)), DSPN) with a characteristic spectrum in the visible range ( A max 430 nm). The absorbance of the reaction product, DSPN, is linearly proportional to the concentration of DDISA and can be used as a method for the colorimetric determination of DDISA. Reaction of DDISA with a molar excess of α-naphthol was also used as a method for inactivating unreacted DDISA to terminate labeling prior to cell fractionation. 3. 3. [ 125I]DDISA reacts avidly with a variety of basic, neutral and acidic proteins as well as with cell membranes to form an acid-stable covalent azo linkage. 4. 4. Effectiveness of labeling of the surface membrane of intact erythrocytes after incubation with [ 125I]DDISA was assessed by the ratio of 125I incorporated into membrane proteins compared to intracellular proteins. When intact erythrocytes were exposed to [ 125I]DDISA, the optimal labeling of membranes occurred at 37°C after 20 min of incubation time and at a concentration of 10 −4 M [ 125I]DDISA in the incubation media. Under these conditions the ratio

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1 −1 cell water · min −1 for this infinite-trans exit procedure. 2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1 −1 cell water · min −1. 3. 3. The K m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1 −1 cell water · min −1. 4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Lymphocyte Subpopulations in Lymphoproliferative Diseases

In analogy with the data of Henkart and Henkart for normal persons, lymphocytes with parallel tubular structures (PTS or the macrotubular bundle) of patients with Hodgkin’s disease were all found to bear an Fc receptor on their cell membrane (EA-RFC). An explanation for this correlation of EA-RFC and PTS or for the increase of EA-RFC in some patients with Hodgkin’s disease was not yet found. Comparison of groups of 22 normal subjects, 20 patients with Hodgkin’s disease, 20 with chronic lymphatic leukemia, and 20 with non-Hodgkin lymphoma showed the EA-RFC to be increased in the Hodgkin group. They were decreased in CLL patients, while mouse erythrocyte rosette-forming cells and membrane surface immunoglobulins in this group were increased. Surface Ig and EA-RFC varied widely in the non-Hodgkin lymphoma group of patients.

Isolation and characterization of the erythrocyte surface membrane of the smooth dogfish, Mustelus canis

1. 1. The plasma membrane of the dogfish erythrocyte is characterized. 2. 2. Surface membranes were isolated from dogfish red cells. 3. 3. Cells suspended in a hypotonic zinc chloride buffer (0.5 mM ZnCl 2, 5 mM Tris-HCl, pH 8.0) tended to enucleate spontaneously. 4. 4. By gentle homogenization relatively high yields of red cell “ghosts” were formed; these were purified using discontinuous gradients of sucrose and of glycerol solutions. Analyses of protein, carbohydrates and lipids were carried out. 5. 5. There did not appear to be marked overall differences in the composition of the surface membrane of the nucleated dogfish erythrocyte compared to those of other species.