Linked Life Data

7795708

Source:http://linkedlifedata.com/resource/pubmed/id/7795708

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1995-8-3
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
0968-7688
pubmed:author
pubmed:issnType
Print
pubmed:volume
12
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
173-82
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:7795708-4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid, pubmed-meshheading:7795708-4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid, pubmed-meshheading:7795708-Amino Acid Sequence, pubmed-meshheading:7795708-Animals, pubmed-meshheading:7795708-Anion Exchange Protein 1, Erythrocyte, pubmed-meshheading:7795708-Anions, pubmed-meshheading:7795708-Antibodies, pubmed-meshheading:7795708-Chymotrypsin, pubmed-meshheading:7795708-Erythrocyte Membrane, pubmed-meshheading:7795708-Humans, pubmed-meshheading:7795708-Ion Exchange, pubmed-meshheading:7795708-Membrane Proteins, pubmed-meshheading:7795708-Molecular Sequence Data, pubmed-meshheading:7795708-Peptide Fragments, pubmed-meshheading:7795708-Precipitin Tests, pubmed-meshheading:7795708-Protein Conformation, pubmed-meshheading:7795708-Rabbits
pubmed:articleTitle
Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein.
pubmed:affiliation
Department of Medicine, University of Toronto, Ontario, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't