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D N Hebert

Publications and source records attributed to D N Hebert.

7 recordsLinked to original sources
PubMed1992

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.

Binding Sites↗
PubMed1991

Evidence that functional erythrocyte-type glucose transporters are oligomers.

In this study we tested the hypothesis that functional erythrocyte-type glucose transporters (GLUT1) exist as oligomeric complexes by expressing chimeric transporter proteins in Chinese hamster ovary cells harboring endogenous GLUT1 transporters. The chimeric transporters were GLUT1-4c, in which the 29 C-terminal residues of human GLUT1 were replaced by the 30 C-terminal residues of rat skeletal muscle glucose transporter (GLUT4), and GLUT1n-4, containing the N-terminal 199 residues of GLUT1 and the 294 C-terminal residues of GLUT4. Endogenous GLUT1 was quantitatively co-immunoprecipitated by using an anti-GLUT4 C-terminal peptide antibody from detergent extracts of Chinese hamster ovary cells expressing either of the chimeric proteins, as detected by immunoblotting the precipitates with an anti-GLUT1 C-terminal peptide antiserum. No co-immunoprecipitation of native GLUT1 with native GLUT4 from extracts of 3T3-L1 adipocytes, which contain both these transporters, was observed with the same antibody. These data are consistent with the hypothesis that GLUT1 transporters exist as homodimers or higher order oligomers and that a major determinant of oligomerization is located within the first 199 residues of GLUT1.

3T3 Cells↗
PubMed1991

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.

Catalysis↗
PubMed1991

Uniporters and anion antiporters.

The past year has seen a flurry of activity in the area of protein-mediated hexose uniport. Topics of interest covered here include: structure-function studies; the interaction of glucose carriers with glycolytic enzymes; regulation of cell surface glucose-carrier concentrations by insulin and the signalling mechanisms involved; and the role of the glucose-carrier isoform, GLUT2, in pancreatic beta-cell glucose-dependent insulin secretion. Nucleoside uniport and Glu-Asp antiport are also discussed briefly.

Adipose Tissue↗
PubMed1989

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport.

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1986

Direct evidence for ATP modulation of sugar transport in human erythrocyte ghosts.

Sugar transport in human erythrocyte ghosts is modulated by low molecular weight factors present in red cell cytosol that induce an asymmetry in Michaelis and velocity constants for sugar entry and exit (Carruthers, A., and Melchior, D. L. (1983) Biochim. Biophys. Acta 728, 254-266). This study examines the possibility that ATP is the transport-modulating factor. The intracellular factor must satisfy at least three criteria. It must reduce Km and Vmax for sugar efflux from inside-out red cell membrane vesicles. It should increase Km for efflux from red cell ghosts. It should have a molecular weight of less than 10 kDa. These criteria are satisfied by ATP. AMP, ADP, GTP, UTP, and ITP are without effect on sugar transport. The following results support the view that the cytosolic factor is ATP. Red cell lysate (obtained by hypotonic lysis of red cells) is unable to modify transport following dialysis against ATP-free medium. The ability of lysate to modify transport is retained following acid extraction. ATP depletion of acid-extracted lysate by treatment with apyrase results in the loss of transport-modulating potency. Myokinase partly restores both the ATP content and the ability of ATP-depleted (apyrase-treated) lysate to modify transport. Addition of ATP to ATP-depleted lysate mimics the ability to myokinase to restore the transport-modulating potency of lysate. ATP is without effect on the number and molecular size of D-glucose-sensitive cytochalasin B-binding proteins in the red cell membrane. These findings demonstrate that the transport-modulating potency of red cell cytosol is quantitatively accounted for by intracellular ATP which acts to modify the catalytic activity of plasmalemmal transporters.

Adenosine Diphosphate↗