Equilibriums and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport

Equilibrium measurements of the inhibition of CB binding to the glucose transporter reported herein indicate that the ternary complexes of CB transporter with EG, PG, or D-glucose are not formed, and these data are consistent with a model for transport in which the substrate binding site exists alternately at the cytoplasmic and external faces of the membrane.

Abstract

Cytochalasin B (CB), n-propyl beta-D-glucopyranoside (PG), and 4,6-O-ethylidene-D-glucose (EG) are known to bind asymmetrically to the human erythrocyte glucose transporter. The first two compounds bind to the inner (cytoplasmic) surface of the transporter, while the latter binds to the outer surface. Equilibrium measurements of the inhibition of CB binding to the glucose transporter reported herein indicate that the ternary complexes of CB transporter with EG, PG, or D-glucose are not formed. Moreover, measurements of CB binding in the presence of both EG and PG or in the presence of high concentrations of D-glucose show that a ternary complex of transporter and sugars bound simultaneously on both sides of the membrane probably does not occur. Finally, the kinetics of dissociation of radiolabeled CB from the transporter in the presence of CB, glucose, PG, and EG have been determined. With the exception of the case of EG, the kinetics fit a simple scheme of rate-limiting unimolecular dissociation, and in no instance do they suggest the existence of a ternary complex of sugar, CB, and transporter. These data are consistent with a model for transport in which the substrate binding site exists alternately at the cytoplasmic and external faces of the membrane, as the result of protein conformational change.