Simultaneous exchange transport of multiple sugar substrates. Ordinate, fractional equilibration of cell water with extracellular radiolabeled sugar; abscissa, time in minutes (note log scale). Each point represents the mean ± SE of at least 3 separate determinations. A: simultaneous exchange of 2.5 mM 3-O-methylglucose (3MG; •) and 2.5 mM d-glucose (○) in red cell ghosts containing 4 mM intracellular ATP. Curves were computed by nonlinear regression assuming the biexponential form A(1 − ) + (1 − A)(1 − ), where k₁ and k₂ are the observed rate constants for phases 1 and 2, respectively, and A is the fractional component of total uptake described by phase 1. For 3MG, k₁ = 0.53 ± 0.06 min⁻¹, k₂ = 0.03 ± 0.01 min⁻¹, and A = 0.76 ± 0.05. For d-glucose, k₁ = 1.45 ± 0.2 min⁻¹, k₂ = 0.05 ± 0.02 min⁻¹, and A = 0.78 ± 0.05. B: simultaneous exchange of 2.5 mM 2-deoxy-d-glucose (2DG; •) and 2.5 mM d-glucose (○) in red cell ghosts containing 4 mM intracellular ATP, 2.5 mM 2DG, and 2.5 mM d-glucose. Curves were computed by nonlinear regression assuming the biexponential form described in A. For 2DG, k₁ = 0.75 ± 0.1 min⁻¹, k₂ = 0.04 ± 0.03 min⁻¹, and A = 0.73 ± 0.07. For d-glucose, k₁ = 0.93 ± 0.1 min⁻¹, k₂ = 0.04 ± 0.02 min⁻¹, and A = 0.74 ± 0.05.

Anomeric analysis of intracellular [³H]3MG following transport. Ordinate, counts per minute (CPM); abscissa, retention time in minutes. A: chromatograms of intracellular [³H]3MG following net uptake intervals of 30 s (blue) and 5 min (red). B: chromatograms of intracellular [³H]3MG following equilibrium exchange intervals of 30 s (blue) and 5 min (red).

3MG exchange in fresh red blood cells treated with and without 2,4-dinitrophenol (DNP). A: scanning electron micrograph of samples of control red blood cells (left) and DNP-treated red blood cells (right) used in B. Scale bar, 7 μm. B: ordinate, fractional equilibration of cell water with 3MG; abscissa, time in minutes. Exchange transport of 2.5 mM 3MG was measured in fresh red blood cells at 4°C following 10-min treatment with DNP (○) or no treatment (•). Curves drawn through points are computed by nonlinear regression to the forms 1 − e^−kt for DNP-treated cells (dashed line) and A(1 − ) + (1 − A)(1 − ) for control and DNP-treated cells (solid lines). The results are k = 0.13 ± 0.01 min⁻¹ (χ² = 0.018) for DNP-treated cells; A = 0.74 ± 0.07, k₁ = 0.41 ± 0.05 min⁻¹, and k₂ = 0.037 ± 0.01 min⁻¹ (χ² = 0.018) for untreated cells; and A = 0.97 ± 0.03, k₁ = 0.141 ± 0.009 min⁻¹, and k₂ = 0.006 ± 0.006 min⁻¹ (χ² = 0.019) for DNP-treated cells. Each point represents the mean ± SE of at least 3 separate determinations.

Effect of mutarotase on d-glucose exchange kinetics. A: ordinate, fractional equilibration of cell water with extracellular radiolabeled sugar; abscissa, time in minutes (note log scale). Transport of 2.5 mM d-glucose was measured without (•) or with (○) 500 U/ml mutarotase resealed inside ghosts containing 4 mM ATP. Each point represents the mean ± SE of at least 3 separate determinations. Dashed curves were drawn by using nonlinear regression assuming the biexponential form described in the legend to Fig. 1. When mutarotase is absent (dashed curve at far right), A = 0.71 ± 0.05, k₁ = 1.4 ± 0.2 min⁻¹, and k₂ = 0.08 ± 0.03 min⁻¹. When mutarotase is present (dashed curve at far left), A = 0.71 ± 0.06, k₁ = 1.3 ± 0.2 min⁻¹, and k₂ = 0.11 ± 0.03 min⁻¹. Transport was simulated using Eqs. 1 and 5, and anomerization was simulated using Eqs. 8a and 8b. Transport parameters were K_α = 50 mM, K_β = 6.3 mM, V_α = 3 mM min⁻¹, and V_β = 5 mM min⁻¹. Anomerization constants are (proceeding from right to left for solid curves; see inset for detail): a, 0 mutarotase, k_obs = 0.005 min ⁻¹ inside and outside; b, mutarotase (500 U/ml) present outside at 10% activity, k_obs(out) = 0.852 min⁻¹, k_obs(in) = 0.005 min⁻¹; c, mutarotase present outside at full activity, k_obs(out) = 8.52 min⁻¹, k_obs(in) = 0.005 min⁻¹; d, mutarotase active both outside and inside at 10% activity, k_obs(out) = k_obs(in) = 0.852 min⁻¹; e, mutarotase fully active outside and 10% active inside, k_obs(out) = 8.52 min⁻¹, k_obs(in) = 0.852 min⁻¹; and f, mutarotase fully active both outside and inside, k_obs(out) = k_obs(in) = 8.52 min⁻¹. B: ordinate, change in refractive index; abscissa, retention time in minutes. HPLC chromatograms are shown of 2 mM α-d-glucose 1 h after dissolution in ice-cold kaline. The conditions were as follows: a, control d-glucose medium not exposed to ghosts (contained 13% β-sugar); b, extracellular d-glucose after incubation with ghosts resealed without exogenous mutarotase (contained 18% β-sugar); c, extracellular d-glucose after incubation with extensively washed ghosts resealed with 500 U/ml mutarotase and exposed to 10 μM cytochalasin B (CCB) plus 100 μM phloretin (contained 26% β-sugar); and d, extracellular d-glucose after incubation with extensively washed ghosts resealed with 500 U/ml mutarotase (contained 64% β-sugar).

Inhibition of 3MG equilibrium exchange transport. Ordinate, fractional equilibration of cell water with extracellular radiolabeled 3MG; abscissa, time in minutes (note log scale). Each point represents the mean ± SE of at least 3 separate determinations. A: inhibition by intra- and extracellular inhibitors of 2.5 mM 3MG exchange in ghosts resealed with 4 mM ATP. Control (solid circles) exchange was biphasic, consistent with the filling of 2 compartments. In the presence of 10 μM CCB (shaded triangles), an endofacial transport inhibitor, transport remained biphasic and both phases of transport were inhibited nearly equally. Transport in the presence of 100 μM phloretin (open circles), an extracellular binding inhibitor was biphasic but only the fast phase was inhibited. Curves drawn through the points were computed by nonlinear regression assuming the biexponential form described in the legend to Fig. 1. Fit constants are shown in Table 5. B: cytochalasin D (CCD) modulation of 2.5 mM 3MG exchange in ghosts resealed with 4 mM ATP. Control (solid circles) exchange was biphasic, consistent with the filling of 2 compartments. In the presence of 10 μM CCD (open circles), transport remained biphasic but the slow phase of transport was inhibited (see Table 5 for fit constants).

A model for ATP-dependent biphasic equilibrium exchange sugar transport in erythrocytes. A: schematic view of the erythrocyte glucose transport protein (GLUT1) translocation pathway. Exo- and endofacial sugar binding sites (e2 and e1, respectively) are connected by an intersite cavity. Reversible flows of sugar (G) between binding sites and between binding sites and bulk solvent are indicated by the appropriate rate constants. When sugar occupies e1, e2, and the central cavity (c), sugars may exchange more rapidly between bound and free states (k_ex). B: simulated (curves) and actual (○) exchange transport data (experimental data taken from Ref. 40) in cells containing 4 mM ATP and equilibrated in 2.5 mM 3MG. Ordinate at left, intracellular radiolabeled 3MG (G_i)/extracellular radiolabeled 3MG (G_o); ordinate at right, concentrations of G bound at site 1 (G1), the intersite cavity (Gc), or site 2 (G2) in mM; abscissa, time in seconds. These simulations assume that G_o is 10 μM, [GLUT1] is 10 μM, and the intersite cavity volume is 7.5 × 10⁻²⁵ liter. C: data are presented as described in B except simulating data were obtained from cells lacking ATP. The results of simulations in B and C are summarized in Table 6.