Evidence for allosteric inhibition sites in the glucose carrier of erythrocytes.

2,4-Fluorodinitrobenzene and 2,3-butanedione, which irreversibly inactivate the glucose transfer system of erythrocytes, have been used as probes to determine whether the substrate site and inner and outer sites for reversible inhibitors are located in the same or different regions of the carrier. Inhibitors bound at an inhibition site exposed in the inward-facing but not the outward-facing form of the carrier (cytochalasin B, androstendione and androstandione) protect the transport system against inactivation by 2,4-fluorodinitrobenzene. Inhibitors bound at an external inhibition site (phloretin) and substrates bound at the transfer site do not protect. In contrast inactivation by 2,3-butanedione is slightly accelerated by internally bound inhibitors, while substrates and substrate analogs bound at the transfer site protect the system. It is shown that fluorodinitrobenzene reacts in the inner inhibition site and butanedione in the substrate site; and further that these sites may be separate binding areas in the carrier linked by allosteric interaction. The consequence of this linkage is that binding of a ligand at the substrate site precludes binding of another ligand at the internal or external inhibition site.