Cooperative nucleotide binding to the human erythrocyte sugar transporter
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology; Department of Molecular Genetics and Microbiology
Medical Subject Headings
3-O-Methylglucose; Adenosine Triphosphate; Azides; Binding Sites; Biological Transport, Active; Erythrocyte Membrane; Glucose Transporter Type 1; Humans; Hydrogen-Ion Concentration; Models, Chemical; Monosaccharide Transport Proteins; Photoaffinity Labels; Proteolipids; Spectrometry, Fluorescence
Life Sciences | Medicine and Health Sciences
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.
Rights and Permissions
Citation: Biochemistry. 2002 Oct 22;41(42):12639-51.