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 membrane skeleton to the lipid bilayer via the bridging protein, ankyrin. To understand how band 3 subunit stoichiometry influences this membrane-skeletal junction, we have induced changes in the band 3 association equilibrium and assayed the kinetics and equilibrium properties of ankyrin binding. We observe that band 3 oligomers convert slowly to dimers and ultimately monomers following removal of ankyrin. Addition of excess ankyrin back to these membranes enriched in dissociated band 3 then shifts band 3 almost entirely to tetramers, confirming that the tetrameric form of band 3 constitutes the preferred oligomeric state of ankyrin binding. 4, 4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) labeling of band 3, which is shown to shift most of the band 3 population to dimers, eliminates the majority of ankyrin-binding sites on the membrane and greatly reduces retention of band 3 in detergent-extracted membrane skeletons. Furthermore, DIDS- modified membranes lack all low affinity ankyrin-binding sites and roughly half of all high affinity sites. Since labeled membranes lack the rapid kinetic phase of ankyrin binding and exhibit only half of the normal amplitude of the slow kinetic phase, it can be concluded that the rapid phase of ankyrin association involves low affinity sites and the slow phase involves high affinity sites. A model accounting for these data and most previous data on ankyrin-band 3 interactions is provided.
Van Dort
H M
HM
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393, USA.
Moriyama
R
R
Low
P S
PS
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000502:physiology; D002918:Chymotrypsin / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D003902:Detergents; D008051:Lipid Bilayers; D002918:Chymotrypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid