Human Erythrocyte Glucose Transport Protein Research Articles

Hydration of AMP and ATP Molecules in Aqueous Solution and Solid Films

Water enables life and plays a critical role in biology. Considered as a versatile and adaptive component of the cell, water engages a wide range of biomolecular interactions. An organism can exist and function only if its self-assembled molecular structures are hydrated. It was shown recently that switching of AMP/ATP binding to the insulin-independent glucose transporter Human Erythrocyte Glucose Transport Protein (GLUT1) may greatly influence the ratio of bulk and bound water during regulation of glucose uptake by red blood cells. In this paper, we present the results on the hydration properties of AMP/ATP obtained by means of dielectric spectroscopy in aqueous solution and for fully ionized forms in solid amorphous films with the help of gravimetric studies.

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

Properties of the Human Erythrocyte Glucose Transport Protein Are Determined by Cellular Context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.

Cooperative nucleotide binding to the human erythrocyte sugar transporter.

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.

Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

Glucose transporter oligomeric structure determines transporter function. Reversible redox-dependent interconversions of tetrameric and dimeric GLUT1.

This study investigates the relationship between human erythrocyte glucose transport protein (GLUT1) oligomeric structure and glucose transporter function. Oligomeric structure was analyzed by hydrodynamic studies of cholate-solubilized GLUT1, by chemical cross-linking studies of membrane-resident GLUT1 and by using conformation-specific antibodies. Transporter function (substrate binding) was analyzed by equilibrium cytochalasin B and D-glucose binding measurements. Erythrocyte-resident glucose transporter is a GLUT1 homotetramer, binds 1 mol of cytochalasin B/2 mol of GLUT1, and presents at least two binding sites to D-glucose. Native structure and function appear to be stabilized by intramolecular disulfide bonds and are preserved during GLUT1 purification by the omission of reductant. Native structure is independent of in vitro and in vivo membrane GLUT1 density but is transformed to dimeric GLUT1 by alkaline reduction. Dimeric GLUT1 binds 1 mol of cytochalasin B/mol of GLUT1, presents a single population of binding sites to D-glucose, and is obtained upon GLUT1 purification in the presence of reductant. Native structure and function are restored by treatment of dimeric GLUT1 with glutathione-disulfide (K0.5 glutathione disulfide = 29 microM). We propose that native structure is established prior to transporter translocation to the plasma membrane and that intrasubunit disulfide bonds promote cooperative subunit interactions that stabilize transporter structure and function.

Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers

The molecular size of purified, human erythrocyte glucose transport protein (GLUT1) solubilized in cholic acid was determined by size-exclusion chromatography (SEC) and sucrose gradient ultracentrifugation. GLUT1 purified in the presence of dithiothreitol (GLUT1 + DTT) is resolved as a complex of average Stokes' radius 5.74 nm by SEC. This complex displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. GLUT1 purified in the absence of dithiothreitol (GLUT1-DTT) is resolved by SEC as at least two particles of average Stokes' radii 5.74 (minor component) and 7.48 nm (major component). Solubilization of GLUT1-DTT in the presence of dithiothreitol reduces the amount of 7.48-nm complex and increases the amount of 5.74-nm complex resolved by SEC. GLUT1-DTT displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. Sucrose gradient ultracentrifugation of GLUT1 + DTT in cholate resolves GLUT1 into two components of 4.8 and 7.6 S. The 4.8S complex is the major component of GLUT1 + DTT. The reverse profile is observed upon sucrose gradient ultracentrifugation of GLUT1-DTT. SEC of human erythrocyte membrane proteins resolves GLUT1 as a major broad peak of average Stokes' radius 7.48 nm and a minor component of 5.74 nm. Both components are characterized by D-glucose-inhibitable cytochalasin B binding. Purified GLUT1 is associated with approximately 26 tightly bound lipid molecules per monomer of transport protein. These data suggest that purified GLUT1 exists as a mixture of homodimers and homotetramers in cholate-lipid micelles and that the presence of reductant during solubilization favors dimer formation.

Identification and characterization of a hepatic microsomal glucose transport protein. T3 of the glucose-6-phosphatase system?

A 52 kDa polypeptide in rat liver microsomes was identified as a glucose-binding protein by its ability to weakly bind cytochalasin B and by its cross-reactivity to an antibody raised against the human erythrocyte glucose transport protein. The microsomal glucose binding polypeptide was purified by affinity chromatography and an antibody was raised against it. The inhibitory effect of this antibody on rat microsomal glucose-6-phosphatase activity and on glucose transport out of microsomal vesicles indicates that this protein is a microsomal glucose transport protein.

Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.

Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein.

Tryptic digestion has been used to investigate the conformational changes associated with substrate translocation by the human erythrocyte glucose transporter. The effects of substrates and inhibitors of transport on the rates of tryptic cleavage at the cytoplasmic surface of the membrane have confirmed previous observations that this protein can adopt at least two conformations. In the presence of phloretin or 4,6-O-ethylidene-D-glucose, the rate of cleavage is slowed. Because these inhibitors bind preferentially at the extracellular surface of the transporter, their effects must result from a conformational change rather than from steric hindrance. A conformational change must also be responsible for the effect of the physiological substrate D-glucose, which is to increase the rate of cleavage. The regions of the protein involved in the conformational changes include both of the large cytoplasmic regions that are cleaved by trypsin: these are the central hydrophilic region of the sequence (residues 213-269) and the hydrophilic C-terminal region (residues 457-492).

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

Membrane-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.

Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein

Conference Article| December 01 1987 Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein ANGELA F. GIBBS; ANGELA F. GIBBS *Departments of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. Search for other works by this author on: This Site PubMed Google Scholar DENNIS CHAPMAN; DENNIS CHAPMAN *Departments of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. Search for other works by this author on: This Site PubMed Google Scholar STEPHEN A. BALDWIN STEPHEN A. BALDWIN †Department of Chemical Pathology and Human Metabolism, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (6): 1128–1129. https://doi.org/10.1042/bst0151128 Article history Received: June 15 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation ANGELA F. GIBBS, DENNIS CHAPMAN, STEPHEN A. BALDWIN; Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein. Biochem Soc Trans 1 December 1987; 15 (6): 1128–1129. doi: https://doi.org/10.1042/bst0151128 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Estradiol stimulation of glucose transport in rat uterus.

Glucose transport in rat uterus was investigated in order to determine whether the stimulation of transport by estradiol was the result of an increase in the amount of glucose transport protein in plasma membranes. In cycling rats, transport was highest during proestrus, the day on which serum estradiol concentrations are highest. In ovariectomized rats, the stimulation of transport was estrogen specific and was nearly complete within 2-3 h after injection of 0.1 microgram estradiol per animal. Estradiol stimulation resulted in a 2- to 3-fold increase in Vmax of 2-deoxy-D-glucose transport with no significant change in Michaelis-Menten constant (Km). Inhibition of the stimulation by cycloheximide or actinomycin D could not be demonstrated, although cycloheximide treatment did cause an increase in the basal rate of transport up to that observed with estradiol treatment. Upon polyacrylamide gel electrophoresis followed by Western blotting, a single protein in uterine plasma membranes reacted with a rabbit antiserum made against purified human erythrocyte glucose transport protein. This protein, believed to be the glucose transport protein, had a mol wt of 49,000 and appeared as a broad band as expected for a glycoprotein. It was found by a protein A-linked immunoassay that the amount of transport protein in uterine plasma membranes was 3.2 +/- 0.4% of the amount in human erythrocyte ghosts. Estradiol treatment had no effect on this value. These data suggest that the stimulation of glucose transport by estradiol occurred by an increase in the intrinsic activity of the transport protein rather than by an increase in the amount of transport protein in plasma membranes.

Isolation and characterization of tryptic fragments from the human erythrocyte glucose-transport protein

Isolation and characterization of tryptic fragments from the human erythrocyte glucose-transport protein

Treatment of the human erythrocyte glucose-transport protein with N-ethylmaleimide

Treatment of the human erythrocyte glucose-transport protein with N-ethylmaleimide