Transmembrane helix-helix interactions and accessibility of H₂DIDS on labelled band 3, the erythrocyte anion exchange protein

Abstract

4,4′-Diisothiocyanodihydrostilbene-2,2′-disulphonate (H₂DIDS), a bifunctional Inhibitor of anion exchange in erythrocytes, reacts with Lys-539 in band 3 at neutral pH and crosslinks to Lys-851 at alkaline pH. The accessibility of H₂DIDS-labelled band 3 was determined using an anti-H₂DIDS antibody and proteolysis. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that a polyclonal antibody raised against H₂DIDS-labelled keyhole limpet hemocyanin bound a variety of stilbene disulphonates in the following order of affinities, H₂DIDS having the highest affinity: H₂DIDS > 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS) > 4-acetamido-4′-isothiocyanostilbene-2,2 disulphonate (SITS) >4,4-dinitrostilbene-2,2′-disulphonate (DNDS) > 4,4′-diaminostilbene-2,2′-disulphonate (DADS). The antibody readily detected mono-or bifunctionally H₂DIDS-labelled band 3 and proteolytic fragments on immunoblots. H₂DIDS attached to Lys-539 is retained in a 7 5 kDa membrane-associated peptide after papain treatment of ghost membranes while the sequence around Lys-851 is more accessible. The band 3 proteolytic fragments protected by the membrane from proteolysis remained associated as a specific complex with a Stokes radius slightly smaller than the dimeric membrane domain after solubilization in detergent solution and retained 82% of the amino acid content of the membrane domain. Circular dichrolsm (CD) measurements of this H₂DIDS-labelled complex showed that it had a very high helical content (86%). The loops connecting the transmembrane segments in H₂DIDS-labelled band 3 are therefore not required to maintain transmembrane helix-helix interactions. Denatured band 3 prelabelled with H₂DIDS was more readily immunoprecipitated with the anti-H₂DIDS antibody than was native band 3 in detergent solution. Deglycosylation of band 3 or proteolytic cleavage of the extramembranous loops did not enhance immunoprecipitation of H₂DIDS-labelled band 3. The stilbene disulphonate inhibitor site is therefore relatively Inaccessible and is bound by a bundle of helices in the native band 3 protein.

Key Words:

• Band 3
• anion transporter
• stilbene disulphonate
• transmembrane helices
• anti-DIDS antibodies