Heterogeneity of aggregates of the human erythrocyte membrane glycoproteins.

The crude red cell glycoproteins obtained by four different methods are compared. The most selective isolation of the major membrane glycoprotein (MN blood group glycoprotein) is achieved by the phenol-water extraction procedure. A different degree of aggregation of this glycoprotein in water and in various buffers is demonstrated. The gel filtration of the crude glycoprotein on a Sepharose 4B column in 0.05 M pyridine--acetate buffer of pH 5.3 gives four fractions with different chemical compostition and serologic properties. The fractions obtained are heterogenous in SDS-PAGE and represent different kinds of glycoprotein aggregates. The distribution of serologic activities in the fractions obtained indicates that A, B and I activities found in the crude prepration of red cell glycoprotein are not connected with MN glycoprotein.     

Aging of the erythrocyte. XII. Protein composition of the membrane

The composition of membrane proteins was compared in erythrocyte fractions of different mean age by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and crossed immunoelectrophoresis. An increase in the content of highmolecular-weight protein aggregates with cell aging was confirmed, and enhanced binding of some hemolysate proteins to the membranes in older cells was found.     

Amphotericin B induced structural changes of the erythrocyte membrane.

Incubation of human erythrocytes in isotonic NaCl-sucrose medium with a relative high concentration of amphotericin B results in the occurrence of distinct structures on the membrane faces as visible by freeze-fracturing. Such kind of structures is known to be formed by phase separation of the lipid components (domain formation). The induction of phase separation is considered to be an additional effect of amphotericin B.     

Electron microscopic toluidine blue staining of the erythrocyte membrane.

Incubation in ferricyanide medium of toluidine blue stained erythrocytes produces opaque precipitates lining either aspect of the cell membrane. In addition, granular and needle-shaped precipitates occupy an intra- or extracellular position. Staining of the outer aspect of the membrane may reflect toluidine blue binding of the erythrocyte glycocalyx. Precipitates at the inner leaflet resemble Ca2+ affinity sites which were considered phosphorylated spectrins. Comparison with the light microscopic topo-optical toluidine blue reaction proved most of the electron dense precitates noncontributive to anisotropic staining.     

Bilirubin-induced erythrocyte membrane cytotoxicity

The kinetics of bilirubin erythrocyte interaction have been followed by scanning electron microscopy. Bilirubin-induced erythrocyte cytotoxicity embodies the interaction of the bile pigment with the outer half of the erythrocyte plasma membrane bilayer couple. This interaction leads to crenation. This membrane event appears to be primary and precedes hemolysis. The membrane crenation is dependent on the concentration of the bile pigment and is reversed by bovine serum albumin again in a concentration-dependent manner. Phospholipids do not alter bilirubin erythrocyte ineraction. Erythrocytes from jaundiced neonates show crenated surface structure in scanning electron microscopy. The crenation depends upon severity of jaundice in neonates. This suggests similarity between in vivo and in vitro mechanisms of cytotoxicity mediated by the bile pigment. Further, phototherapy reverses the process of membrane crenation. The in vivo photocatabolities isolated from urine of jaundiced neonates are nontoxic to erythrocyte membrane.     

Ganglioside and rabbit erythrocyte membrane receptor for staphylococcal alpha-toxin.

The hemolytic activity of staphylococcal alpha-toxin is inhibited by an N-acetylglucosamine-containing ganglioside (GlcNAc-ganglioside) but not by any of the related glycolipids. The GlcNAc-ganglioside also is precipitated with the toxin by a gel-diffusion technique. It is postulated that GlcNAc-ganglioside may be the membrane receptor for the alpha-toxin.     

The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane.

The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.     

Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes.

Erythrocytes containing mature gametocytes of Plasmodium falciparum circulate in the blood until they are ingested by a mosquito, an event that triggers gametogenesis and lysis of the infected erythrocyte. It was previously shown that a parasite protein (Pf155/RESA) accumulates in the erythrocyte cytoplasm next to the parasitophorous vacuolar membrane (S. Uni, A. Masuda, M. J. Stewart, R. Nussenzweig, and M. Aikawa, Am. J. Trop. Med. Hyg., 36:481-488, 1987). Using a monoclonal antibody to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we have studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after triggering gametogenesis, the parasitophorous membrane no longer surrounded the parasite, bringing the parasite membrane in contact with the erythrocyte cytoplasm. Clear spaces appeared throughout the hemoglobin-rich host cytoplasm; Pf155/RESA was now localized in the cytoplasm directly surrounding the spaces. No membrane existed between the spaces and the erythrocyte cytoplasm. The spaces with surrounding Pf155/RESA protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane (15 min after triggering gametogenesis), the protein was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane. The mechanism by which Pf155/RESA remained aggregated around the spaces and its role in erythrocyte lysis are unknown. It is of interest that the parasite appeared to use the same molecule during invasion of erythrocytes and during release of gametes from infected erythrocytes.     

Influence of pH on elastic deformability of the human erythrocyte membrane.

Fresh human blood was diluted 1:5000 in buffered saline-sucrose solution and titrated to a pH varying from 4.5 to 10.5 with 0.1 N HCl or 0.1 N NaOH. Circular regions of the membrane of individual cells were then deformed at 25 degrees C by aspiration into a micropipette having an internal tip diameter of 0.9-1.4 micron. A membrane surface elasticity modulus, mu (dyn/cm), was computed from the relationship between length of the aspirated membrane and the deforming pressure according to a two-dimensional membrane model. Surface elasticity increases with decreasing pH and with time after the cell suspension is acidified, rising several orders of magnitude with a t1/2 of 1--5 h as pH is lowered from 7.2 to 4.6. This increase in mu is only partially reversible. pH greater than 7.2 had little effect on mu. Membrane surface elasticity is not affected by variations in external [Ca2+] over the range of 0--50 mM, tonicity of the suspension medium from 275--400 mosM, or age of 0--50 h. Addition of 50 mM NaHCO3 to the medium increases the rate of change of mu at a given pH. These results suggest that the elastic properties of the red cell membrane are largely determined by interactions among structural proteins located on the cytoplasmic surface of the membrane and that these interactions are initiated by changes in intracellular pH.     

Digitonin induced alterations of the erythrocyte membrane as visible by freeze-fracturing.

Treatment of erythrocytes by low concentrations of digitonin results in the formation of elongated, bulged membrane areas free from particles and intramembraneous tubular structures emerged from these domains. At higher concentrations the tubular structures are present also outside of the membrane. A second, not bulged type of particle-free areas is more or less rounded, but mostly of rectangular shape. The rate of this type of domains increases with the concentration of digitonin. Breaks in these areas lead to a formation of sheets which results in a breakdown of the membrane structure finally into a brittle mass of many small sheets lying irregularly one upon the other. Already during the first steps of this membrane breakdown hemolysis takes place. Cross-linking of the proteins with glutaraldehyde does not entirely block up the membrane alterations. In fixed erythrocytes the formation fo elongated domains is restricted, no tubular structures are present and no dislocations of particles can be observed. Nevertheless smooth sheets localized also outside of the membrane are formed. However the investigated ghosts are not stabilized by glutaraldehyde against the effects of digitonin. Particle dislocations as well as all the other membrane alterations are present. The implications of the obtained results are: 1. The elongated domains and the tubular structures are probably not digitonin-cholesterol-complexes. 2. The formation of crystalline regions by digitonin-cholesterol-complexes destroys the membrane structure.     

Aging of the erythrocyte. IX. Fluorescence studies on changes in membrane properties

Fluorescence studies of membranes prepared from density-separated red blood cells demonstrated an increase in lipid viscosity (judging from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 1-aminonaphthalane-8-sulfonate and changes in the physical state of proteins (judging from tryptophan fluorescence intensity and polarization). These alterations may be partly due to lipid peroxidation. In vivo accumulation of products of this process in the membranes was confirmed. Changes were also found in the distribution pattern of cell populations of different mean age in a cell sorter, probably due to alterations in rheological properties of the erythrocytes.     

Phosphorylation of human erythrocyte membrane protein. Differences according to the assay procedure.

Phosphorylation of the human erythrocyte membrane proteins by gamma (32P) ATP was studied at pH 6 and pH 7.4, at 30 degrees C, with incubation times varying from 5 to 90 minutes, and with or without cyclic AMP. Incorporated radioactivity was much higher at pH 7.4 because of the prevalent activity of cAMP independent protein-kinase. Maximum incorporation was obtained in both pH after 30-45 minutes incubation, thereafter incorporated radioactivity was either stable or decreased. The part of the radioactivity due to cAMP stimulation was low and seems constant with the incubation time. Analysis of the substrates showed the predominant cAMP independent protein kinase activity in the phosphorylation of the spectrin second component and component III and that of cAMP dependent activity in the phosphorylation of component II4, IV5 and other minor bands.     

Extracellular cations and the movement of choline across the erythrocyte membrane

1. The ability of human erythrocytes to accumulate choline is abolished when external Na is replaced by Cs, Rb, K or Li but is increased when the external cation is Mg or Ca.2. The unidirectional influx of choline is reduced when external Na is replaced by other monovalent cations but is not changed when Na is replaced by Mg or Ca.3. The unidirectional efflux of choline into a choline-free medium is increased when external Na is replaced by other monovalent cations and markedly reduced when Na is replaced by Mg or Ca. When the external medium contains 1 mM choline, changing the external cation has virtually no effect on the rate of choline efflux.4. When the extracellular concentrations of K and Na are similar to that found in the intracellular water, choline appears to become passively distributed across the cell membrane; when the extracellular K is then replaced by Cs a net efflux of choline against a concentration gradient results.5. It is concluded that the choline carrier may be described as a cation carrier with a high affinity for choline and affinities for Cs > Rb > K > Li > Na and that these monovalent cations can cross the membrane on the choline transport system.