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11	Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes Gottschalk, Ingo January 2002 (has links) <p>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. </p><p>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 <i>in vitro</i> and <i>in vivo</i>. </p><p>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.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p> Biochemistry Affinity Binding Biomembrane Biotin Chromatography Cytochalasin B Dissociation constant Drug absorption Equilibrium Glucose GLUT1 Immobilization Immobilized liposome chromatography Interaction Liposome Membrane protein Membrane vesicle Partitioning Phospholipid bilayer Proteoliposome Quantitative Red blood cell Solute Specific Streptavidin Wheat germ agglutinin Biokemi Biochemistry Biokemi
12	Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter Lagerquist Hägglund, Christine January 2003 (has links) <p>The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.</p><p>Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.</p> Biochemistry Affinity Aromatic amino acids Binding Biomembrane Biotin Chromatography Cytochalasin B Dihydrocytochalasin B Dissociation constant Drug absorption Ethanol Equilibrium Glucose GLUT1 Immobilization Immobilized liposome chromatography Interaction Liposome Membrane protein Membrane vesicle Partitioning Phospholipid bilayer Proteoliposome Quantitative Red blood cell Specific Streptavidin Tyrosine Tryptophan Biokemi Biochemistry Biokemi
13	Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter Lagerquist Hägglund, Christine January 2003 (has links) The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods. Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested. Biochemistry Affinity Aromatic amino acids Binding Biomembrane Biotin Chromatography Cytochalasin B Dihydrocytochalasin B Dissociation constant Drug absorption Ethanol Equilibrium Glucose GLUT1 Immobilization Immobilized liposome chromatography Interaction Liposome Membrane protein Membrane vesicle Partitioning Phospholipid bilayer Proteoliposome Quantitative Red blood cell Specific Streptavidin Tyrosine Tryptophan Biokemi Biochemistry Biokemi
14	Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes Gottschalk, Ingo January 2002 (has links) 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. Biochemistry Affinity Binding Biomembrane Biotin Chromatography Cytochalasin B Dissociation constant Drug absorption Equilibrium Glucose GLUT1 Immobilization Immobilized liposome chromatography Interaction Liposome Membrane protein Membrane vesicle Partitioning Phospholipid bilayer Proteoliposome Quantitative Red blood cell Solute Specific Streptavidin Wheat germ agglutinin Biokemi Biochemistry Biokemi

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