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Immobilised biomembrane affinity chromatography for binding studies of membrane proteins
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
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2002 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 768, no 1, 31-40 p.Article, review/survey (Refereed) Published
Abstract [en]

Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly.

Place, publisher, year, edition, pages
2002. Vol. 768, no 1, 31-40 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90003DOI: 10.1016/S0378-4347(01)00483-2PubMedID: 11939556OAI: oai:DiVA.org:uu-90003DiVA: diva2:162003
Available from: 2002-10-03 Created: 2002-10-03 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
Open this publication in new window or tab >>Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction.

Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo.

Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.

An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 43 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 755
Keyword
Biochemistry, Affinity, Binding, Biomembrane, Biotin, Chromatography, Cytochalasin B, Dissociation constant, Drug absorption, Equilibrium, Glucose, GLUT1, Immobilization, Immobilized biomembrane affinity chromatography, Immobilized liposome chromatography, Interaction, Liposome, Membrane protein, Membrane vesicle, Partitioning, Phospholipid bilayer, Proteoliposome, Quantitative, Red blood cell, Solute, Specific, Streptavidin, Wheat germ agglutinin, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-2668 (URN)91-554-5413-5 (ISBN)
Public defence
2002-10-25, lecture hall B41, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-10-03 Created: 2002-10-03 Last updated: 2013-06-12Bibliographically approved

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