title: Cooperative nucleotide binding to the human erythrocyte sugar transporter.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Lazare Research Building, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA.
authors.forename: Erin K
authors.initials: EK
authors.lastname: Cloherty
authors.forename: Kara B
authors.initials: KB
authors.lastname: Levine
authors.forename: Christopher
authors.initials: C
authors.lastname: Graybill
authors.forename: Anthony
authors.initials: A
authors.lastname: Carruthers
mesh_terms: D019325:3-O-Methylglucose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D001386:Azides; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D019920:Photoaffinity Labels; D011510:Proteolipids; C495677:SLC2A1 protein, human; C020485:proteoliposomes; D019325:3-O-Methylglucose; C017040:8-azidoadenosine 5'-triphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate