Biomaterial Database

Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein. 1995

C Landolt-Marticorena, and J R Casey, and R A Reithmeier

Department of Medicine, University of Toronto, Ontario, Canada.

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.

UI	MeSH Term	Description	Entries
D007474	Ion Exchange	Reversible chemical reaction between a solid, often one of the ION EXCHANGE RESINS, and a fluid whereby ions may be exchanged from one substance to another. This technique is used in water purification, in research, and in industry.	Exchange, Ion
D008565	Membrane Proteins	Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.	Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011233	Precipitin Tests	Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate.	Precipitin Test,Test, Precipitin,Tests, Precipitin
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011817	Rabbits	A burrowing plant-eating mammal with hind limbs that are longer than its fore limbs. It belongs to the family Leporidae of the order Lagomorpha, and in contrast to hares, possesses 22 instead of 24 pairs of chromosomes.	Belgian Hare,New Zealand Rabbit,New Zealand Rabbits,New Zealand White Rabbit,Rabbit,Rabbit, Domestic,Chinchilla Rabbits,NZW Rabbits,New Zealand White Rabbits,Oryctolagus cuniculus,Chinchilla Rabbit,Domestic Rabbit,Domestic Rabbits,Hare, Belgian,NZW Rabbit,Rabbit, Chinchilla,Rabbit, NZW,Rabbit, New Zealand,Rabbits, Chinchilla,Rabbits, Domestic,Rabbits, NZW,Rabbits, New Zealand,Zealand Rabbit, New,Zealand Rabbits, New,cuniculus, Oryctolagus
D002918	Chymotrypsin	A serine endopeptidase secreted by the pancreas as its zymogen, CHYMOTRYPSINOGEN and carried in the pancreatic juice to the duodenum where it is activated by TRYPSIN. It selectively cleaves aromatic amino acids on the carboxyl side.	Alpha-Chymotrypsin Choay,Alphacutanée,Avazyme
D004910	Erythrocyte Membrane	The semi-permeable outer structure of a red blood cell. It is known as a red cell 'ghost' after HEMOLYSIS.	Erythrocyte Ghost,Red Cell Cytoskeleton,Red Cell Ghost,Erythrocyte Cytoskeleton,Cytoskeleton, Erythrocyte,Cytoskeleton, Red Cell,Erythrocyte Cytoskeletons,Erythrocyte Ghosts,Erythrocyte Membranes,Ghost, Erythrocyte,Ghost, Red Cell,Membrane, Erythrocyte,Red Cell Cytoskeletons,Red Cell Ghosts
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man