Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

1988 ◽  
Vol 66 (10) ◽  
pp. 1126-1133 ◽  
Author(s):  
Elena Burdett ◽  
Amira Klip
Keyword(s):  
Metabolic Regulation ◽  
Glucose Transporter ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Glucose Transporters ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Western Blots ◽  
Intact Cells ◽  
Human Erythrocyte Membranes

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55 000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze–thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters. It is concluded that the antibody may be reacting with exofacial regions of the glucose transporter in intact cells. This suggests that the antibodies may be used to detect relative changes in glucose transporter number on the cell surface.

scholarly journals Inhibition by nucleosides of glucose-transport activity in human erythrocytes

1988 ◽  
Vol 249 (2) ◽  
pp. 383-389 ◽  
Author(s):  
S M Jarvis
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Binding Activity ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Transport Activity ◽  
Intact Cells ◽  
Order Of Magnitude ◽  
Glucose Carrier

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.

Sulfhydryl substituents of the human erythrocyte hexose transport mechanism

1986 ◽  
Vol 250 (6) ◽  
pp. C853-C860 ◽  
Author(s):  
R. E. Abbott ◽  
D. Schachter ◽  
E. R. Batt ◽  
M. Flamm
Keyword(s):  
Human Erythrocyte ◽  
Transport Mechanism ◽  
Cytochalasin B ◽  
Erythrocyte Membranes ◽  
Type I ◽  
Type Ii ◽  
Hexose Transport ◽  
Intact Cells ◽  
Type Iii ◽  
Human Erythrocyte Membranes

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.

scholarly journals Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids

1990 ◽  
Vol 265 (2) ◽  
pp. 471-477 ◽  
Author(s):  
M J Watts ◽  
J R Dankert ◽  
B P Morgan
Keyword(s):  
Cerebrospinal Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Isolation And Characterization ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.

scholarly journals Expression of the major sialoglycoprotein (glycophorin) on erythroid cells in human bone marrow

Blood ◽  
1978 ◽  
Vol 52 (2) ◽  
pp. 379-387 ◽  
Author(s):  
CG Gahmberg ◽  
M Jokinen ◽  
LC Andersson
Keyword(s):  
Bone Marrow ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Human Erythrocyte ◽  
Bone Marrow Cells ◽  
Protein A ◽  
Human Bone ◽  
Erythrocyte Membranes ◽  
Erythroid Cells ◽  
Human Erythrocyte Membranes

The major sialoglycoprotein of human erythrocyte membranes (glycophorin) is one of the most-studied membrane proteins. Although the structure is relatively well known, almost nothing is known about its expression in erythroid cells. To study this we raised an antiserum that reacted specifically with this protein. This was accomplished by immunization of rabbits with a preparation of glycophorin followed by absorption with En(a-) erythrocyte membranes, which lack glycophorin. By use of this antiserum and a staphylococcus protein A technique we could establish that only bone marrow cells of erythrocyte lineage express glycophorin at the cell surface. This occurs in basophilic normoblasts and later stages of erythrocyte differentiation, whereas pronormoblasts do not seem to contain glycophorin.

scholarly journals Reconstitution studies of amino acid transport system L in rat erythrocytes

1993 ◽  
Vol 292 (3) ◽  
pp. 655-660 ◽  
Author(s):  
S Y M Yao ◽  
R George ◽  
J D Young
Keyword(s):  
Amino Acid ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Phospholipid Vesicles ◽  
Transport Activity ◽  
Protein Ratio ◽  
Intact Cells ◽  
Amino Acid Transport System ◽  
Simple Diffusion ◽  
System L

In many cell types, including human erythrocytes, membrane transport of hydrophobic amino acids such as leucine and phenylalanine is mediated primarily by Na(+)-independent system L. In this paper we demonstrate that erythrocytes from the rat have a 400-fold higher system L transport capacity than human erythrocytes. We have exploited this high transport activity to achieve the first successful reconstitution of an erythrocyte amino acid transporter into phospholipid vesicles. Rat erythrocyte membranes were depleted of extrinsic membrane proteins, solubilized in 50 mM n-octyl glucoside and reconstituted into egg-yolk phospholipid vesicles by a gel filtration freeze-thaw protocol. Optimal reconstitution of transport activity occurred at lipid/protein ratios of 25-35:1. At a lipid/protein ratio of 25:1, one-half of the total uptake of L-[14C]leucine (0.2 mM, 25 degrees C) was inhibited by 2 mM phloretin and thus judged to be carrier-mediated. This component of L-leucine uptake was inhibited by non-radioactive L-phenylalanine and L-leucine, and only to a very much weaker extent by glycine and L-alanine. Two other inhibitors of system L in intact cells, MK196 and PCMBS (p-chloromercuriphenylsulphonate), were also effective inhibitors of phloretin-sensitive L-leucine transport in reconstituted proteoliposomes. Phloretin-insensitive uptake of L-leucine in proteoliposomes occurred by simple diffusion across the lipid bilayer.

scholarly journals Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

2012 ◽  
Vol 302 (3) ◽  
pp. H553-H559 ◽  
Author(s):  
Meera Sridharan ◽  
Elizabeth A. Bowles ◽  
Jennifer P. Richards ◽  
Medina Krantic ◽  
Katie L. Davis ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Atp Release ◽  
Anion Channel ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Voltage Dependent Anion Channel ◽  
Pannexin 1 ◽  
Ip Receptor ◽  
Voltage Dependent ◽  
Human Erythrocyte Membranes

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (Gi or Gs), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-xL BH44–23 and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-xL BH44–23 attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.

Evidence against the Involvement of Mutarotase in the D-Glucose Uptake Activity of Isolated Human Erythrocyte Membranes

1972 ◽  
Vol 50 (9) ◽  
pp. 1028-1030 ◽  
Author(s):  
Arthur Kahlenberg ◽  
Gary Miller
Keyword(s):  
Glucose Uptake ◽  
Membrane Transport ◽  
Glucose Transport ◽  
Human Erythrocyte ◽  
Transport Process ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Uptake Activity ◽  
Glucose Carrier

Mutarotase, the enzyme catalyzing the interconversion of the anomeric forms of D-glucose, has recently been suggested to be the membrane glucose carrier in human erythrocytes. However, hemoglobin-free human erythrocyte membranes possessing D-glucose uptake activity were found to be free of mutarotase activity. Mutarotase activity was detected in the membrane-free hemolysates of the cells. It is therefore concluded that the D-glucose uptake activity of isolated erythrocyte membranes is not due to the binding of the sugar to mutarotase, and that this enzyme is not involved in glucose transport in a manner compatible with most presently held concepts of the membrane transport process.

scholarly journals The human erythrocyte nucleoside and glucose transporters are both band 4.5 membrane polypeptides

1983 ◽  
Vol 214 (3) ◽  
pp. 995-997 ◽  
Author(s):  
J S R Wu ◽  
S M Jarvis ◽  
J D Young
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporters ◽  
Erythrocyte Membranes ◽  
Nucleoside Transporter ◽  
Hexose Transport ◽  
Major Site ◽  
Equilibrium Binding ◽  
Human Erythrocyte Membranes

Human erythrocyte membranes and partially purified nucleoside transporter (band 4.5 and 7) were photoaffinity labelled with 3H-labelled 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine under equilibrium binding conditions. Band 4.5 was the major site of radiolabelling in both preparations. These experiments provide additional evidence to implicate band 4.5 polypeptides in nucleoside permeation, proteins previously shown to be involved in hexose transport.

scholarly journals Labelling of the intramembranous region of the major sialoglycoprotein of human erythrocytes with a photosensitive hydrophobic probe

1979 ◽  
Vol 179 (2) ◽  
pp. 265-272 ◽  
Author(s):  
E Wells ◽  
J B Findlay
Keyword(s):  
Membrane Proteins ◽  
Human Erythrocyte ◽  
Human Erythrocytes ◽  
Labelling Pattern ◽  
Erythrocyte Membranes ◽  
Transmembrane Region ◽  
Human Erythrocyte Membranes ◽  
Hydrophobic Probe

Human erythrocyte membranes were incubated with the photosensitive hydrophobic reagent 1-azido-r-iodo[3H]benzene and the mixture was irradiated. The major sialoglycoprotein was then isolated and the labelled polypeptide subjected to proteolytic dissection. Characterization of the purified tryptic and chymotryptic peptides show that the probe is covalently attached only to the transmembrane region of the protein. This labelling pattern is discussed in relation to the use of such reagents for the identification of segments of membrane proteins exposed to the hydrophobic millieu of the membrane.

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