Richard E. Abbott

Surface adsorption of dextrans on human red cell membrane

The adsorption isotherms of dextrans with molecular weights of 40,000 (Dx 40), 74,000 (Dx 70), and 450,000 (Dx 500) were studied on normal red blood cells (RBCs) and RBCs with surface charge depleted by neuraminidase treatment. The adsorption curves on neuraminidase-treated RBCs showed a two-step behavior with secondary adsorption commencing at a bulk concentration of approximately 5 g/100 ml. A plateau adsorption of approximately 9 × 10−14 g/RBC was attained with bulk concentrations between 12 and 20 g/100 ml. Normal RBCs showed similar adsorption curves as neuraminidase-treated RBCs in Dx 70, as well as in Dx 500, with bulk concentrations up to 5 g/100 ml. Further increases in bulk concentration of Dx 70 or Dx 500 caused greater adsorption on normal RBCs than on neuraminidase-treated RBCs, with a plateau concentration of approximately 15 × 10−14 g/RBC. Adsorption of Dx 40 on normal RBCs was higher than that on neuraminidase-treated RBCs at all bulk concentrations. These results on surface adsorption have been correlated with the aggregation behavior of the same cell systems. The data offer evidence in support of the hypothesis that surface adsorption of dextrans leads to RBC aggregation by bridging adjacent cell surfaces. The results also indicate that the adsorption of dextrans to RBC surface is a dynamic, reversible process in which the adsorbed molecules exchange readily with the molecules in the bulk or those attached to another cell surface. RBC aggregation is associated with an apparent decrease in surface adsorption as the opposing cell surfaces in the aggregate share their adsorption sites via the same dextran molecules.

Effects of benzyl alcohol on erythrocyte shape membrane hemileaflet fluidity and membrane viscoelasticity

The effects of benzyl alcohol on cell shape, hemileaflet lipid fluidity and membrane rheology of human red blood cells were studied. Membrane fluidity was assessed by determining the fluorescence anisotropy of permeant probes (1,6-diphenyl-1,3,5-hexatriene,12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate) and a new impermeant probe (N-stachyosylsuccinic acid dihydrazide-2-(9-anthroyloxy)stearate). Measurements made on intact red blood cells reflected primarily the outer leaflet fluidity while measurements made on red blood cells ghosts reflected the fluidity of both leaflets. Membrane viscoelasticity was determined by micropipette aspiration. Treatment of intact red blood cells with benzyl alcohol up to 50 mM caused progressive stomatocytic shape change but no change in membrane viscoelasticity, 1,6-diphenyl-1,3,5-hexatriene anisotropy or stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time; similar treatment of leaky ghosts yielded decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time. With benzyl alcohol above 50-60 mM, intact red blood cells became echinocytic, and decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time occurred in both intact cells and ghosts; there was no change in membrane viscoelasticity. These results indicate that benzyl alcohol up to 50 mM affects primarily the inner leaflet of the red blood cell membrane and that higher concentrations affect both leaflets. These increases in membrane fluidity are not associated with changes in membrane viscoelasticity. This study illustrates the use of fluorescence techniques to monitor specifically the lipid fluidity of each hemileaflet of the erythrocyte membrane.

LIPID FLUIDITY OF THE INDIVIDUAL HEMILEAFLETS OF HUMAN ERYTHROCYTE MEMBRANES

The impermeant fluorescent probes (MIMAR reagents) described here permit the assessment of the lipid fluidity of individual membrane hemileaflets. They should also prove useful for examining the outer hemileaflets of the plasma membranes of intact cells. The observations, thus far, that normal human erythrocyte membranes have a characteristic asymmetry of fluidity, with the outer leaflet more fluid, correspond to prior findings with Mycoplasma, Newcastle Disease viral envelopes, and mouse LM cells. Hence, it is possible that the pattern is quite general in biological membranes. The particular lipid and protein components of the human-erythrocyte membrane that underly the fluidity asymmetry are unknown. The increased content of phosphatidylcholine in the outer leaflet and of the anionic phospholipids in the inner leaflet would be consonant with the fluidity difference. On the other hand, sphingomyelin, which tends to decrease fluidity, is localized mainly in the outer leaflet. Unknown at present is whether the cholesterol content of the two leaflets differs. From the results reported above, it is tempting to speculate that exogenously added cholesterol tends to localize in the outer leaflet, normally the more fluid leaflet, whereas endogenous cholesterol is more readily removed from the inner leaflet. This suggests, but clearly does not establish, that in the normal erythrocyte the cholesterol content of the inner leaflet exceeds that of the outer. Lastly, integral membrane proteins are expected to decrease lipid fluidity, and the usual pattern seen on freeze-fracture of large numbers of intra-membranous particles on the cytoplasmic face may signify a greater influence of protein in the inner leaflet. The hypothesis that perturbations of the fluidity of a given hemileaflet influence the membrane proteins (and their associated functions) in that leaflet is well-supported by the evidence described above. On the other hand, we understand less well the mechanisms by which lipid fluidity influences the proteins. For example, the decrease in sulfhydryl group reactivity of spectrin, actin, and Band 3 owing to cholesterol depletion (Table 7) may be due to a physical displacement of these proteins, as suggested by Borochov and Shinitzky. Why then does the reactivity of glyceraldehyde-phosphate dehydrogenase sulfhydryl groups increase under these conditions? There remains much to learn about membrane molecular mechanics and lipid-protein interactions. In such studies the impermeant MIMAR probes described here should prove useful.

Topography and functions of sulfhydryl groups of the human erythrocyte glucose transport mechanism

SummaryMembrane-impermeant and -permeant maleimides were applied to characterize the location and function of the sulfhydryl (SH) groups essential for the facilitated diffusion mediated by the human erythrocyte glucose transport protein. Three such classes have been identified. Type I SH is accessible to membrane-impermeant reagents at the outer (exofacial) surface of the intact erythrocyte. Alkylation of this class inhibits glucose transport; D-glucose and cytochalasin B protect against the alkylation. Type II SH is located at the inner (endofacial) surface of the membrane and is accessible to the membrane-impermeant reagent glutathione maleimide only after lysis of the erythrocyte. D-glucose enhances, while cytochalasin B reduces, the alkylation of Type II SH by maleimides. Reaction of Types I and II SH with an impermeant maleimide increases the half-saturation concentration for binding of D-glucose to erythrocyte membranes. By contrast, inactivation of Type III SH markedly decreases the half-saturation concentration for the binding of D-glucose and other transported sugars. Type III SH is inactivated by the relatively lipid-soluble reagents N-ethylmaleimide (NEM) and dipyridyl disulfide, but not by the impermeant glutathione maleimide. Type III SH is thus located in a hydrophobic membrane domain. A kinetic model constructed to explain these observations indicates that Type III SH is required for the translocation event in a hydrophobic membrane domain which leads to the dissociation of glucose bound to transport sites at the membrane surfaces.

The viscoelastic properties of monolayers of red cell membrane proteins

Abstract The rheological properties of spectrin-actin (S + A) surface films have been studied from the point of view of their possible role in the red cell membrane structure and function. Our observations indicate the following: (1) The amount of S + A that is normally present in the membrane can form at least one monolayer and probably two when one considers that other membrane components (e.g., band 3 protein) are intermixed with it; (2) the viscoelastic properties of S + A films vary with the film thickness and the composition of the adjacent phases; (3) The magnitudes of the rheological parameters of the films are close to the analogous parameters of the red cell membrane and thus may account for these properties in the intact red cell.

Erythrocyte membrane proteins: A modified gorter-grendel experiment

SummaryThe pressure-area isotherm and shear resistance of spectrin-actin monolayers indicate a close-packed structure at about 1.0 m2/mg protein. This surface area is equivalent to a thickness of about two monolayers at the erythrocyte membrane inner face. The maximum elasticity (lowest compressibility) occurs at 0.7 m2/mg protein, indicating the limit of reversible compression. The mechanical properties of the monolayers approximate those of the intact membrane, suggesting that the structures are similar and that these monolayers may account for many of thein vivo properties.