Testing transport systems for competition between pairs of reversible inhibitors. Inhibition of erythrocyte glucose transport by cytochalasin B and steroids.

When two different inhibitors, which are unrelated in structure, inhibit transport by binding to the carrier on the same side of the membrane, it is important for an understanding of the mechanism to decide whether they add to a single common site or to two separate sites. To help resolve this problem, kinetic tests are described for determining whether the inhibitors compete with one another. The test involves measuring the inhibition produced by the inhibitors when acting alone or together. The rate observed with both inhibitors present is then compared with that predicted from the separate inhibitions, for the case of either one or two inhibition sites. The test is applied to the inhibition of glucose transport in erythrocytes by cytochalasin B and the steroids androstenedione and androstanedione. These inhibitors were previously shown to bind to sites which are distinct from the substrate site and which are only present in the inward-facing form of the carrier. Cytochalasin B is now found to compete with the steroids. This finding, together with the earlier evidence that both inhibitors sterically protect the carrier against reaction with fluorodinitrobenzene, leads us to conclude that a single site binds both types of inhibitor.