Steady state kinetics of erythrocyte anion exchange. Evidence for site-site interactions.
Steady state velocities for the rate of exchange of extracellular dithionite for intracellular sulfate were measured in the presence of single and pairs of cis inhibitors. The purpose of these kinetic studies was to test the current two site model used to explain the nonhyperbolic transport kinetics characteristic of the erythrocyte anion exchange system. This model suggests the existence of a transport site and an external, pure noncompetitive inhibitory modifier site, both of which are thought to exist on each subunit of the band 3 protein dimer. Our results show negative cooperative transport curves when the velocity is plotted versus the external anion concentration at constant trans anion concentration. Reversible inhibition by SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate) caused the degree of negative cooperativity to change nonlinearly as a function of SITS concentration. This nonlinear dependence was such that SITS actually increased the calculated degree of negative cooperativity and then decreased it suggesting site-site interactions. Multiple inhibition studies confirm the existence of site-site interactions by showing that SITS and acetate are nonmutually exclusive inhibitors with negative interactions or mutually hindered inhibitor binding. These results are not consistent with the transport site-modifier site kinetic model in its original form since that model proposes only one pathway for transport with no effect of one inhibitor on the binding of the other. Possible kinetic models are discussed in an attempt to explain the present results and to relate them to what is currently known about the structure of band 3 protein.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D004227:Dithionite / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013431:Sulfates / Q000097:blood
D000838:Anions; D013431:Sulfates; D004227:Dithionite; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid