scholarly journals Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex

2014 ◽  
Vol 306 (10) ◽  
pp. C910-C917 ◽  
Author(s):  
Jay M. Sage ◽  
Anthony Carruthers
Keyword(s):  
Steady State ◽  
Vitamin C ◽  
Blood Cells ◽  
Dehydroascorbic Acid ◽  
Membrane Vesicles ◽  
Human Erythrocytes ◽  
Cytoplasmic Surface ◽  
Glucose Transport Protein ◽  
Inside Out ◽  
Primary Substrate

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3- O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l−1·min−1, respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex.

Membrane orientation of sheep spectrin

1980 ◽  
Vol 58 (10) ◽  
pp. 1120-1130
Author(s):  
P. Prokopchuk ◽  
A. U. Sargent
Keyword(s):  
Sheep Erythrocyte ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Variable Degree ◽  
Vertebrate Species ◽  
Membrane Orientation ◽  
Cytoplasmic Surface ◽  
Immunofluorescence Studies ◽  
Surface Labelling ◽  
Inside Out

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membranes localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected i0mmunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.

scholarly journals Osmotic Vesicle Collapse of Sealed Inside–Out Membrane Vesicles From Red Blood Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Teresa Tiffert ◽  
Virgilio L. Lew
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Blood Cells ◽  
Membrane Vesicles ◽  
Membrane Transporters ◽  
Plasma Membrane Vesicles ◽  
Functional Studies ◽  
Transmission Electron ◽  
Proper Interpretation ◽  
Inside Out

The preparation of plasma membrane vesicles from a large variety of cells has contributed a wealth of information on the identity and vectorial properties of membrane transporters and enzymes. Vesicles from red blood cell (RBC) membranes are generated in media of extremely low tonicity. For functional studies, it is required to suspend the vesicles in higher tonicity media in order to bring the concentrations of the substrates of transporters and enzymes under investigation within the physiological ranges. We investigated the effects of hypertonic transitions on the vesicle morphology using transmission electron microscopy. The results show that hypertonic transitions cause an irreversible osmotic collapse of sealed membrane vesicles. Awareness of the collapsed condition of vesicles during functional studies is critical for the proper interpretation of experimental results.

scholarly journals Lysine-691 of the anion exchanger from human erythrocytes is located on its cytoplasmic surface

1998 ◽  
Vol 336 (2) ◽  
pp. 443-449 ◽  
Author(s):  
Hans K. ERICKSON ◽  
Jack KYTE
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Liquid Scintillation ◽  
Pyridoxal Phosphate ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Differential Centrifugation ◽  
Cytoplasmic Surface ◽  
Inside Out

A combination of vectorial modification and site-directed immunochemistry has been used to determine the disposition, with respect to the membrane, of Lys-691 of the anion exchanger from human erythrocytes. Intact erythrocytes and inside-out vesicles were vectorially modified in the same container with pyridoxal phosphate and sodium [3H]borohydride. The modified inside-out vesicles were separated from erythrocytes by differential centrifugation and the vesicles and erythrocyte membranes were treated with alkali and digested with trypsin and thermolysin to liberate the peptides IVSKPER and IVSK{Nε-[4´-(5´-phospho-[4-3H]pyridoxyl)]}PER. These peptides, containing the unmodified and modified versions of Lys-691, were retrieved from the digests by site-directed immunochemistry and were identified by HPLC and liquid scintillation spectroscopy. Both the inside-out vesicles and the intact erythrocytes contained the peptide IVSKPER, however, the 3H-label from the phosphopyridoxylated peptide could be detected only in the inside-out vesicles. The incorporation of 3H into Lys-691 of the anion exchanger from inside-out vesicles was at least 30-fold greater than the incorporation into Lys-691 of the anion exchanger from intact erythrocytes. It follows that Lys-691 of the anion exchanger is located on the cytoplasmic surface of the plasma membrane.

Role of monosaccharide transporter in vitamin C uptake by placental membrane vesicles

1986 ◽  
Vol 250 (4) ◽  
pp. C637-C641 ◽  
Author(s):  
R. L. Ingermann ◽  
L. Stankova ◽  
R. H. Bigley
Keyword(s):  
Vitamin C ◽  
Concentration Gradient ◽  
Cytochalasin B ◽  
Dehydroascorbic Acid ◽  
Membrane Vesicles ◽  
Sodium Concentration ◽  
Reduced Form ◽  
High Concentration ◽  
Simple Diffusion ◽  
Monosaccharide Transporter

Dehydroascorbic acid (DHA), the reversibly oxidized form of vitamin C, was taken up much more rapidly than L-glucose into membrane vesicles prepared from the maternal face of the human placental syncytiotrophoblast. DHA uptake was sensitive to inhibition by cytochalasin B and was independent of a sodium concentration gradient. At equilibrium, the concentration of DHA in the vesicles did not exceed that of the medium. DHA and the D-glucose analogue, 3-O-methyl-D-glucose (3-O-MG) appeared to compete with one another for the transporter. The 3-O-MG and DHA inhibitory constants were indistinguishable. Vesicles loaded with a high concentration of 3-O-MG and suspended in low 3-O-MG displayed a marked, transitory enhancement of DHA, but not L-glucose uptake. These findings suggest that DHA is taken into the first cellular boundary of the placenta between maternal and fetal circulations by the sodium-independent monosaccharide transporter. In contrast to DHA, L-ascorbic acid, the reversibly reduced form of vitamin C, was taken into these vesicles much more slowly. This uptake was not affected by cytochalasin B nor by a sodium concentration gradient; it appeared to occur by simple diffusion.

scholarly journals The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 197 (2) ◽  
pp. 283-291 ◽  
Author(s):  
A Graham
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Labelling Pattern ◽  
F1 Atpase ◽  
Crossed Immunoelectrophoresis ◽  
Cytoplasmic Surface ◽  
Membrane Bound ◽  
Inside Out

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.

scholarly journals Characteristics of Chloride Transport in Human Red Blood Cells

1973 ◽  
Vol 61 (2) ◽  
pp. 185-206 ◽  
Author(s):  
Robert B. Gunn ◽  
Mads Dalmark ◽  
D. C. Tosteson ◽  
J. O. Wieth
Keyword(s):  
Steady State ◽  
Blood Cells ◽  
Transport Mechanism ◽  
Chloride Transport ◽  
Human Erythrocytes ◽  
Hydrogen Ions ◽  
Chloride Flux ◽  
Human Red Blood Cells ◽  
Functional Forms ◽  
Exchange Flux

The efflux of chloride-36 from human erythrocytes under steady-state conditions is a saturable process that is competitively inhibited by bicarbonate and noncompetitively inhibited by acetate. This chloride self-exchange flux is reversibly dependent on the pH of the medium between 5.7 and 9.6 with a maximum flux at pH 7.8. The increase in chloride flux between pH 5.7 and 7.8 is inexplicable by the fixed charge hypothesis. The interpretations are made that chloride transport in human erythrocytes is carrier mediated, that bicarbonate utilizes the same transport mechanism, and that the mechanism can be titrated with hydrogen ions into less functional forms for chloride transport.

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