EFFECT OF BAND-3 SUBUNIT EQUILIBRIUM ON THE KINETICS AND AFFINITY OF ANKYRIN BINDING TO ERYTHROCYTE-MEMBRANE VESICLES

The membrane-spanning protein, band 3, anchors the spectrin-based memb rane skeleton to the lipid bilayer via the bridging protein, ankyrin, To understand how band 3 subunit stoichiometry influences this membran e-skeletal junction, we have induced changes in the band 3 association equilibrium and assayed the kinetics and equilibrium properties of an kyrin binding. We observe that band 3 oligomers convert slowly to dime rs and ultimately monomers following removal of ankyrin. Addition of e xcess ankyrin back to these membranes enriched in dissociated band 3 t hen shifts band 3 almost entirely to tetramers, confirming that the te trameric form of band 3 constitutes the preferred oligomeric state of ankyrin binding. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DID S) labeling of band 3, which is shown to shift most of the band 3 popu lation to dimers, eliminates the majority of ankyrin-binding sites on the membrane and greatly reduces retention of band 3 in detergent-extr acted membrane skeletons. Furthermore, DIDS- modified membranes lack a ll low affinity ankyrin-binding sites and roughly half of all high aff inity sites. Since labeled membranes lack the rapid kinetic phase of a nkyrin binding and exhibit only half of the normal amplitude of the sl ow kinetic phase, it can be concluded that the rapid phase of ankyrin association involves low affinity sites and the slow phase involves hi gh affinity sites. A model accounting for these data and most previous data on ankyrin-band 3 interactions is provided.