title: 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 dichroism (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.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Landolt-Marticorena
authors.forename: J R
authors.initials: JR
authors.lastname: Casey
authors.forename: R A
authors.initials: RA
authors.lastname: Reithmeier
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000032:analysis / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D000838:Anions / Q000378:metabolism; D000906:Antibodies / Q000737:chemistry; D002918:Chymotrypsin / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007474:Ion Exchange; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D011817:Rabbits
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D000906:Antibodies; D008565:Membrane Proteins; D010446:Peptide Fragments; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002918:Chymotrypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid