scholarly journals Spectroscopic assays for measuring quantities of erythrocyte membrane "halves".

1982 ◽  
Vol 92 (1) ◽  
pp. 44-52 ◽  
Author(s):  
K A Fisher
Keyword(s):  
Membrane Lipids ◽  
Membrane Surface ◽  
Erythrocyte Membranes ◽  
Splitting Technique ◽  
Cell Monolayers ◽  
Human Erythrocyte Membranes ◽  
Glass Cell ◽  
Extracellular Side ◽  
Lower Ratio ◽  
Cytoplasmic Side

The quantities of outer and inner"halves" produced by freeze-fracturing human erythrocyte membranes have been measured by visible and fluorescence spectroscopy. Assays have been developed that are based on the use of two membrane surface markers: hemoglobin (Hb), a native marker for the cytoplasmic side of the membrane, and fluoresceinated concanavalin A (FITC-Con-A), a marker for the extracellular side. Hb absorbance is proportional to the fraction of cytoplasmic "half" membranes, and FITC fluorescence is proportional to the fraction of extracellular "halves." A procedure is described for the preparation of surface-labeled, intact erythrocytes suitable for the formation of homogeneous, planar cell monolayers of square-centimeter dimensions on polylysine-treated glass (PL-glass). Cell monolayers were frozen and fractured, and the fractions of absorbance and fluorescence in each of the two split portions determined. The PL-glass portion of membrane contained a substantially higher ratio of fluorescence to absorbance than unsplit controls, and its paired portion, a complementary lower ratio, demonstrating that the PL-glass portion was significantly enriched in extracellular "half" membrane. Experiments investigating split membrane recovery show that the double labeled membrane splitting technique is well suited to analysis of the transmembrane distribution of membrane lipids and polypeptides using methods that do not require quantitation by electron microscopy.

scholarly journals Cytosolic protein binding to band-3 protein inhibits endocytosis of isolated human erythrocyte membranes

1982 ◽  
Vol 207 (3) ◽  
pp. 595-598 ◽  
Author(s):  
K A Cordes ◽  
J M Salhany
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Band 3 ◽  
Erythrocyte Membranes ◽  
Band 3 Protein ◽  
High Affinity ◽  
Human Erythrocyte Membranes ◽  
Affinity Interaction ◽  
Cytoplasmic Side

Recent studies of haemoglobin binding to the cytoplasmic side of the erythrocyte membrane have shown that the predominant high-affinity interaction occurs with the major integral membrane protein known as band-3 protein and that this interaction may occur within the intact erythrocyte in a manner regulated by cell pH. We report here that haemoglobin and glyceraldehyde 3-phosphate dehydrogenase binding to band-3 protein in isolated membranes can inhibit endocytosis during vesiculation in vitro. The specificity of this effect was demonstrated by showing that myoglobin, which has an affinity for the membrane fully one to two orders of magnitude lower than that for haemoglobin, does not inhibit endocytosis.

scholarly journals ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES

1973 ◽  
Vol 59 (2) ◽  
pp. 395-406 ◽  
Author(s):  
Garth L. Nicolson ◽  
Richard G. Painter
Keyword(s):  
Iron Hydroxide ◽  
Membrane Surface ◽  
Erythrocyte Membranes ◽  
Temperature Dependent ◽  
Colloidal Iron ◽  
Acetylneuraminic Acid ◽  
Peripheral Protein ◽  
Human Erythrocyte Membranes ◽  
Γ Globulins ◽  
Topographic Distribution

The effects of affinity-purified antispectrin γ-globulins on the topographic distribution of anionic residues on human erythrocytes membranes was investigated using collo ida iron hydroxide labeling of mounted, fixed, ghost membranes. Antispectrin γ-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37°C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (<0.05 mg/ml) or high (>5–10 mg/ml concentrations of sequestered antispectrin was dispersed, but the distribution at intermediate concentrations (0.1–5 mg/ml) was highly aggregated. The aggregation of colloidal iron hydroxide binding sites was time and temperature dependent and required the sequestering of cross-linking antibodies (antispectrin Fab could not substitute for γ-globulin antibodies) inside the ghosts. Prior glutaraldehyde fixation or fixation at the time of hemolysis in antispectrin solutions prevented the antispectrin-induced colloidal iron site aggregation. The antispectrin reacted exclusively at the inner ghost membrane surface and the colloidal iron hydroxide bound to N-acetylneuraminic acid residues on the outer membrane surface which are overwhelming on the sialoglycoprotein glycophorin. These results were interpreted as evidence for a structural transmembrane linkage between the inner surface peripheral protein spectrin and the integral membrane component glycophorin.

scholarly journals Presence in Human Erythrocyte Membranes of a Novel Form of Sialidase Acting Optimally at Neutral pH

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 2047-2056 ◽  
Author(s):  
Bruno Venerando ◽  
Amelia Fiorilli ◽  
Gian Luigi Croci ◽  
Guido Tettamanti
Keyword(s):  
Membrane Surface ◽  
Erythrocyte Membranes ◽  
Ph Optimum ◽  
Sialidase Activity ◽  
Human Erythrocyte Membranes ◽  
Resealed Ghosts ◽  
Triton X ◽  
Inside Out ◽  
Physiologic Role ◽  
Optimal Ph

Abstract The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37°C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.

scholarly journals Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes

1987 ◽  
Vol 248 (1) ◽  
pp. 21-26 ◽  
Author(s):  
H Sato ◽  
S Aono ◽  
R Semba ◽  
S Kashiwamata
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Erythrocyte Membranes ◽  
Binding Properties ◽  
Before And After ◽  
Human Erythrocyte Membranes ◽  
Bilirubin Binding ◽  
The Right

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

scholarly journals Biological Activity of Blackcurrant Extracts (Ribes nigrumL.) in Relation to Erythrocyte Membranes

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Dorota Bonarska-Kujawa ◽  
Sylwia Cyboran ◽  
Romuald Żyłka ◽  
Jan Oszmiański ◽  
Halina Kleszczyńska
Keyword(s):  
Antioxidant Activity ◽  
Free Radicals ◽  
Erythrocyte Membrane ◽  
Membrane Lipids ◽  
Membrane Surface ◽  
Antioxidant Properties ◽  
Erythrocyte Membranes ◽  
Leaf Extracts ◽  
Hydrophobic Region ◽  
Spectrophotometric Methods

Compounds contained in fruits and leaves of blackcurrant (Ribes nigrumL.) are known as agents acting preventively and therapeutically on the organism. The HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and flavonoids in leaves, that have antioxidant activity and are beneficial for health. The aim of the research was to determine the effect of blackcurrant fruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant properties. The effect of the extracts on osmotic resistance, shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined with spectrophotometric methods. The FTIR investigation showed that extracts modify the erythrocyte membrane and protect it against free radicals induced by UV radiation. The results show that the extracts do not induce hemolysis and even protect erythrocytes against the harmful action of UVC radiation, while slightly strengthening the membrane and inducing echinocytes. The compounds contained in the extracts do not penetrate into the hydrophobic region, but bind to the membrane surface inducing small changes in the packing arrangement of the polar head groups of membrane lipids. The extracts have a high antioxidant activity. Their presence on the surface of the erythrocyte membrane entails protection against free radicals.

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312 ◽  
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

Abstract This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

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