Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer

Abstract

(1) The Michaelis-Menten parameters for hexose transfer in erythrocytes, erythrocyte ghosts and inside-out vesicles at 20 degrees C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392-403). (2) The external Km for infinite-cis exit of D-glucose in cells and ghosts is 3.6 +/- 0.5 mM. (3) Dilution of cellular solute (up to X 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the Vm for net D-glucose exit. The Km for net exit, however, falls from 32.4 +/- 3.7 mM in intact cells to 12.9 +/- 2.3 mM in ghosts. This effect is reversible. (4) Infinite-cis net D-glucose uptake measurements in cells and ghosts reveal the presence of a low Km, high affinity internal site of 5.9 +/- 0.8 mM. The Vm for net glucose entry increases from 23.2 +/- 3.7 mmol/1 per min in intact cells to 55.4 +/- 6.3 mmol/l per min in ghosts. (5) The external Km for infinite-cis D-glucose exit in inside-out vesicles is 6.8 +/- 2.7 mM. The kinetics of zero-trans D-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the Km and Vmax for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, Vmax for zero-trans exit is reduced; Km for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces Vmax for D-glucose exit but is without effect on the external Km for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.