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Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads
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, Surface Biotechnology.
2003 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 784, no 1, 203-208 p.Article in journal (Refereed) Published
Abstract [en]

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin–agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70±14 nM for CB and 12±3 mM for glucose binding to GLUT1, are similar to those reported earlier.

Place, publisher, year, edition, pages
2003. Vol. 784, no 1, 203-208 p.
Keyword [en]
Wheat germ agglutinin, Cytochalasin B, GLUT1, Immobilization, Superporous agarose
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90004DOI: 10.1016/S1570-0232(02)00758-4PubMedID: 12504199OAI: oai:DiVA.org:uu-90004DiVA: diva2:162004
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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