The human erythrocyte sugar transporter is also a nucleotide binding protein.
We have previously shown that ATP interacts with an intracellular, stereoselective, regulatory site(s) on the human erythrocyte sugar transport system to modify transport function in a hydrolysis-independent manner. This present study examines the nucleotide binding properties of the human erythrocyte sugar transport system. We demonstrate by transport studies in ghosts, by nucleotide binding studies with purified transport protein by measurements of nucleotide inhibition of 8-azidoadenosine 5'-[gamma-32P]triphosphate (azido-ATP) photoincorporation into purified carrier, and by analysis of nucleotide inhibition of carboxyl-terminal peptide antisera binding to purified glucose carrier than the glucose transport protein binds (with increasing order of affinity) AMP, ADP, ATP, 5'-adenylyl imidodiphosphate (AMP-PNP), and 1,N6-ethenoadenosine 5'-triphosphate (EATP) at a single site. The carrier lacks detectable ATPase activity and GTP binding capacity. While AMP and ADP bind to the carrier protein and act as competitive inhibitors of ATP binding, these nucleotides are unable to mimic the ability of ATP, AMP-PNP, and EATP to modify the catalytic properties of the sugar transport system. Limited tryptic digestion of azido-ATP-photolabeled carrier suggests that the region of the glucose transport protein containing the intracellular cytochalasin B binding and extracellular bis(mannose) binding domains [residues 270-456; Holman, G. D., & Rees, W. D. (1987) Biochim. Biophys. Acta 897, 395-405] may also contain the intracellular ATP binding site.
Carruthers
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Department of Biochemistry, University of Massachusetts Medical School, Worcester 01655.
Helgerson
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D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D001386:Azides / Q000378:metabolism; D001693:Biological Transport, Active; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*
D001386:Azides; D009004:Monosaccharide Transport Proteins; D000249:Adenosine Monophosphate; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D005947:Glucose