Net sugar transport is a multistep process. Evidence for cytosolic sugar binding sites in erythrocytes

Abstract

Human erythrocyte net sugar transport is hypothesized to be rate-limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules [Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249]. The present study examines these hypotheses. Protein-mediated 3-O-methylglucose uptake at 4 degrees C by human erythrocytes and by resealed, hypotonically lysed erythrocytes (ghosts) is inhibited by increasing solvent viscosity. Protein-mediated transport and transbilayer diffusion of the slowly transported substrate 6-NBD glucosamine are unaffected by increasing solvent viscosity. These findings suggest that protein-mediated 3-O-methylglucose transport is diffusion-limited in erythrocytes. More detailed analyses of red cell 3-O-methylglucose uptake (at 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling of a small compartment (C1 = 29 +/- 6% total cell volume; k1 = 7.4 +/- 1.7 min-1) and slow filling of a larger compartment (C2 = 71 +/- 6% total cell volume k2 = 0.56 +/- 0.11 min-1). Erythrocyte D-glucose net uptake at 4 degrees C is also a biphasic process. Transmembrane sugar leakage is a monoexponential process indicating that multicomponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limiting 3-O-methylglucose concentrations is described by single exponential kinetics. This demonstrates that multicomponent sugar uptake does not result from influx into two populations of cells with widely different sugar transporter content. We conclude that biexponential sugar uptake results from slow (relative to transport) exchange of sugars between serial, intracellular sugar compartments. Biexponential sugar uptake is observed under equilibrium exchange conditions (intracellular sugar concentration = extracellular sugar concentration) but only at 3-O-methylglucose concentrations of less than 1 mM. Above this sugar concentration, exchange uptake is a monoexponential process. Because diffusion rates are independent of diffusant concentration, this suggests that multicomponent uptake results from high-affinity sugar binding within the cell. The concentration of cytosolic binding sites (30 microM, Kd(app) = 400 microM) was estimated from the equilibrium cellular 3-O-methylglucose space. Biexponential net 3-O-methylglucose uptake is also observed in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure to hemin. This demonstrates that neither membrane-bound nor free cytosolic hemoglobin forms the sugar-binding complex.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP-dependent sugar transport complexity in human erythrocytes

“…Table 1 summarizes fit parameters for ghosts lacking and containing ATP. Equilibrium exchange time courses have been studied previously by this laboratory, but biphasic transport was observed only at the lowest 3MG concentration employed (0.1 mM) (23), where biphasic exchange may be related to GLUT1 sugar binding. That study did not monitor transport beyond 5 min, which explains why biphasic equilibrium exchange transport at higher 3MG concentrations was not observed.…”

“…Evidence for an unstirred layer comes from three sources. Net 3MG uptake in human red blood cells displays biphasic kinetics, suggesting rapid equilibration with an unstirred layer and slower equilibration with bulk cytosol (23). Steady-state sugar exit in human red blood cells deviates from Michaelis-Menten kinetics at low intracellular sugar concentration in a way that is characteristic of an intracellular unstirred layer (10,28).…”

Kinetics of glucose transport and sequestration in lactating bovine mammary glands measured in vivo with a paired indicator/nutrient dilution technique

“…The estimated r v for glucose of 0.34 is thus only 17% of the expected ratio and likely represents the size of the intermediate occlusion compartment. Occlusion volume was estimated at 30 -40% of intracellular glucose space in human erythrocytes (6,12) and 35% in cultured bovine mammary epithelial cells (25). Intracellular ATP will influence the size of the occlusion space, possibly through allosteric interaction with the GLUT1 homotetramer.…”

Cytokine Regulation of Facilitated Glucose Transport in Human Articular Chondrocytes

“…Glucose serves as a main energy source in chondrocytes (4,5), as a main precursor for glycosaminoglycan synthesis (6, 7), and as a regulator of the cell responses to certain growth factors (44). GLUTs also participate in facilitated transport of glucosamine and N-acetylglucosamine (45,46), which can modify inflammatory responses of chondrocytes (47) and serve as efficient precursors of glycosaminoglycan synthesis (48). Finally, GLUTs (predominantly GLUT1 and GLUT3) facilitate transport of dehydroascorbic acid (49), which induces chondrocyte differentiation (50) and regulates extracellular matrix gene expression, including type II collagen (51).…”

“…It has been demonstrated that GLUT1 protein has a single N-glycosylation site at Asn 45 , which is heterogeneously glycosylated (25). Glycosylated GLUT1 protein was shown to have a 2-to 2.5-fold lower K m for 2DG binding (62), an increased protein stability, and an increased rate of protein incorporation into the plasma membrane than its nonglycosylated analog (62).…”