Evidence for two disulfide bonds important to the functioning of the renal outer cortical brush-border membrane...

The effects of the disulfide reducing agent dithiothreitol on the renal outer cortical brushborder membrane D-glucose transporter are studied. Two apparently independent results of dithiothreitol treatment are observed. The first is a completely reversible increase in the affinity of the carrier for the nontransported competitive inhibitor phlorizin. The second is a partially reversible loss of phlorizin binding sites. Similar results are observed with the monothiol disulfide reducing agent beta-mercaptoethanol. Reversal of both effects of dithiothreitol could be prevented by exposing the membranes to an excess of N-ethylmaleimide following treatment with the reducing agent. When transported sugars are included with dithiothreitol treatment, the magnitude of both of its effects on the carrier are influenced; more specifically, the affinity for phlorizin is further increased and the loss of phlorizin binding sites is reduced. Nontransported sugars are without effect. These observations provide strong evidence for the existence of (at least) two disulfide bonds important to the functioning of this transporter.