Initial steps of alpha- and beta-D-glucose binding to intact red cell membrane

The kinetics of the initial phases of D-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable L-glucose has no kinetic response, in contrast to D-glucose kinetics. Other controls show that D-glucose binding is inhibited by cytochalasin B and by extracellular D-maltose. A typical time course for a transportable sugar, such as D-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of 0.5-100 sec-1 at 20 degrees C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the Kd of the epimer, D-galactose, from the Kd for D-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the alpha- and beta-D-glucose anomers. The exponential 1 activation energy of the beta-anomer, 13.6 +/- 1.4 kcal mol-1, is less than that of alpha-D-glucose, 18.4 +/- 0.8 kcal mol-1, and the two Arrhenius lines cross at approximately 23.5 degrees C. The temperature dependence of the kinetic response following alpha-D-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6 degrees C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.