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Predicate | Object |
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
2
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pubmed:dateCreated |
1995-8-3
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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.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/4,4'-Diisothiocyanostilbene-2,2'-Dis...,
http://linkedlifedata.com/resource/pubmed/chemical/4-Acetamido-4'-isothiocyanatostilben...,
http://linkedlifedata.com/resource/pubmed/chemical/Anion Exchange Protein 1...,
http://linkedlifedata.com/resource/pubmed/chemical/Anions,
http://linkedlifedata.com/resource/pubmed/chemical/Antibodies,
http://linkedlifedata.com/resource/pubmed/chemical/Chymotrypsin,
http://linkedlifedata.com/resource/pubmed/chemical/Membrane Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Peptide Fragments
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pubmed:status |
MEDLINE
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pubmed:issn |
0968-7688
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:volume |
12
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
173-82
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pubmed:dateRevised |
2006-11-15
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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
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pubmed:articleTitle |
Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein.
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pubmed:affiliation |
Department of Medicine, University of Toronto, Ontario, Canada.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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