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ATP-dependent sugar transport complexity in human erythrocytes

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts

Submitted 19 June 2006 ; accepted in final form 15 August 2006

ABSTRACT

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar]_{intracellular} = [sugar]_{extracellular}, where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of 20 µM). Immunofluorescence-activated cell sorting analysis shows that biphasic exchange does not result from heterogeneity in cell size or GLUT1 content. Nucleoside transporter-mediated uridine exchange proceeds as rapidly as 3MG exchange but is monoexponential regardless of cytoplasmic [ATP]. This eliminates cellular heterogeneity or an ATP-dependent, nonspecific intracellular diffusion barrier as causes of biphasic exchange. Red cell ghost 3MG and uridine equilibrium volumes (130 fl) are unaffected by ATP. GLUT1 intrinsic activity is unchanged during rapid and slow phases of 3MG exchange. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional), or chemical (isomerization) barrier to equilibrate with cell water. Partial transport inhibition with the use of cytochalasin B or maltose depresses both rapid and slow phases of transport, thereby eliminating the physical barrier hypothesis. We propose that biphasic 3MG transport results from ATP-dependent, differential transport of 3MG anomers in which V_max/apparent K_m for -3MG exchange transport is 19-fold greater than V_max/apparent K_m for -3MG transport.

carrier-mediated transport; transport kinetics; transport regulation

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