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)
Helgerson
A L
AL
Department of Biochemistry, University of Massachusetts Medical Center, Worcester 01605.
Hebert
D N
DN
Naderi
S
S
Carruthers
A
A
D019325:3-O-Methylglucose; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001693:Biological Transport, Active / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002250:Carbon Radioisotopes; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008563:Membrane Lipids / Q000097:blood; D008757:Methylglucosides / Q000378:metabolism*; D008759:Methylglycosides / Q000378:metabolism*; D011865:Radioisotope Dilution Technique
D002250:Carbon Radioisotopes; D008563:Membrane Lipids; D008757:Methylglucosides; D008759:Methylglycosides; D019325:3-O-Methylglucose; D000001:Calcimycin; D000255:Adenosine Triphosphate; D002118:Calcium