Kinetic evidence for modulation by glycophorin A of a conformational equilibrium between two states of band 3 (SLC4A1) bound reversibly by the competitive inhibitor DIDS.
Recent evidence has suggested that erythrocytes naturally deficient in glycophorin A (GPA) have a reduced V(max) for monovalent anion exchange. Unanswered is whether miss-folding of band 3 during biosynthesis, or the absence of GPA modulation of properly folded band 3 is responsible. Here, I determine the effect of selective depletion of GPA on the kinetics of reversible binding of the competitive transport inhibitor DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) to properly folded band 3. Reversible binding of DIDS follows biphasic kinetics: a fast phase {DIDS+band 3<-->(DIDS-band 3), k(1), k(-1)} and a slower phase {(DIDS-band 3)<-->(DIDS-band 3), k(2), k(-2)}. Selective depletion of GPA was accomplished by pretreating membranes with Triton X-100, over a range where erythrocyte hemolysis is inhibited by the detergent (0% to 0.03%, v/v). Pretreatment with sublytic Triton X-100: (a) virtually completely depleted GPA, (b) did not deplete membrane-bound band 3, and (c) slowed the overall rate of reversible binding of DIDS to band 3. Data analysis and model simulation studies indicated that the decrease in the rate of binding of DIDS was due exclusively to a decrease in k(-2), with no change in the initial rate of binding. Thus, depletion of GPA does not alter the native conformation of band 3 at the DIDS binding site, but rather modulates a conformational equilibrium between two states of the binary complex formed by the competitive inhibitor DIDS, reversibly bound to properly folded band 3.
Salhany
James M
JM
Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-4510, USA.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000187:drug effects; D001667:Binding, Competitive; D003198:Computer Simulation; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects; D006021:Glycophorins / Q000502:physiology*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D025941:Protein Interaction Mapping
D001457:Anion Exchange Protein 1, Erythrocyte; D006021:Glycophorins; D017830:Octoxynol; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid