| 1. |
- Beigi, Farideh, et al.
(författare)
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Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport
- 1998
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Ingår i: International Journal of Pharmaceutics. - 0378-5173 .- 1873-3476. ; 164:1-2, s. 129-137
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Tidskriftsartikel (refereegranskat)abstract
- Drug partitioning into lipid bilayers was studied by chromatography on liposomes and biomembranes immobilized in gel beads by freeze–thawing. The drug retention volume was expressed as a capacity factor, Ks, normalized with respect to the amount of immobilized phospholipid. Log Ks values for positively charged drugs on brain phosphatidylserine (PS)/egg phosphatidylcholine (PC) liposomes decreased as the ionic strength was increased, increased as the PS:PC ratio or the pH was increased and varied linearly with the temperature. Log Ks values for beta-blockers, phenothiazines and benzodiazepines on egg phospholipid (EPL) liposomes correlated well with corresponding values on red cell membrane lipid liposomes (r2=0.96), and on human red cell membrane vesicles containing transmembrane proteins (r2=0.96). A fair correlation was observed between the values on EPL liposomes and those on native membranes of adsorbed red cells (r2=0.86). Compared to the data obtained with liposomes, the retentions of hydrophilic drugs became larger and the range of log Ks values more narrow on the vesicles and the membranes, which expose hydrophilic protein surfaces and oligosaccharides. Lower correlations were observed between drug retention on EPL liposomes and egg PC liposomes; and between retention on liposomes (or vesicles) and immobilized artificial membrane (IAM) monolayers of PC analogues. Absorption of orally administered drugs in humans (literature data) was nearly complete for drugs of log Ks values in the interval 1.2–2.5 on vesicles. Both vesicles and liposomes can thus be used for chromatographic analysis of drug–membrane interaction and prediction of drug absorption.
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| 2. |
- Gottschalk, Ingo, 1971-
(författare)
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Chromatographic Studies of Solute Interactions with Immobilized Red Blood Cells and Biomembranes
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
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| 4. |
- Gottschalk, Ingo, et al.
(författare)
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Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:23, s. 6875-6882
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Tidskriftsartikel (refereegranskat)abstract
- Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.
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| 5. |
- Gottschalk, Ingo, et al.
(författare)
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Immobilised biomembrane affinity chromatography for binding studies of membrane proteins
- 2002
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Ingår i: Journal of chromatography. B. - 1570-0232 .- 1873-376X. ; 768:1, s. 31-40
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 6. |
- Gottschalk, Ingo, et al.
(författare)
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Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads
- 2003
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Ingår i: Journal of chromatography. B. - 1570-0232 .- 1873-376X. ; 784:1, s. 203-208
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Lagerquist Hägglund, Christine, et al.
(författare)
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Cytochalasin B-binding and transport properties of the Glut1 human red cell glucose transporter
- 2000
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Ingår i: Recent research developments in bioenergetics Vol. 1 (2000) Part II. - Trivandrumc : Transworld Research Network. - 818684659X ; , s. 117-129
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Bokkapitel (refereegranskat)abstract
- The mechanism by which the D-glucose transporter Glut1 facilitates transport of monosaccharides, dehydroascorbic acid and possibly nicotinamide across membranes toward lower chemical activity is thought to involve binding of the substrate at the internal or external face of the protein followed by a thermally triggered conformational change that allows release of the substrate from a binding site exposed at the opposite side of the protein. In the presumably tetrameric transporter in the cell membrane, pairs of identical subunits may work in concert. In each dimer, one of the subunits may expose an external substrate-binding site and the other an internal site at a given moment (State 1). Only the internal site binds the inhibitor cytochalasin B. Cytochalasin B-binding analyses and transport data support this mechanism [Hamill, S., Cloherty, E.K., and Carruthers, A. 1999, Biochemistry, 38, 16974]. Both alkaline disulfide reduction of Glut1 in the cell membrane and polylysine-coating of red cells convert Glut1 to a functionally monomeric state that offers a cytochalasin B-site on every subunit (State 2). Cysteine-scanning mutagenesis experiments with transport and inhibition analyses indicate that the proposed transmembrane segments 5 and 7 are amphipathic α-helices that probably line the glucose permeation pathway. The future determination of the 3D structures of sugar transporters may elucidate the transport mechanism and subunit interactions.
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| 8. |
- Lagerquist Hägglund, Christine, et al.
(författare)
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Ethanol weakens cytochalasin B binding to the GLUT1 glucose transporter and drug partitioning into lipid bilayers
- 2004
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Ingår i: Journal of chromatography A. - : Elsevier BV. - 0021-9673. ; 1031, s. 113-116
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Tidskriftsartikel (refereegranskat)abstract
- Ethanol weakens the specific interaction between the human red blood cell (RBC) glucose transporter GLUT1 and the inhibitor cytochalasin B (CB). The chromatographic retention volume of cytochalasin B on stationary phases consisting of GLUT1-containing membranes decreased with increasing ethanol concentration in the eluent. The apparent Kd values for the ethanol-GLUT1 interaction were 0.37, 0.45 and 0.64 M for red blood cells, red blood cell membrane vesicles and proteoliposomes, respectively, all much higher than the Kd values for D-glucose or cytochalasin B interaction with GLUT1. Ethanol also decreased the partitioning of cytochalasin B and drugs into phospholipid bilayers.
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| 9. |
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| 10. |
- Lundahl, Per, et al.
(författare)
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Chromatographic approaches to liposomes, proteoliposomes and biomembrane vesicles
- 1999
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Ingår i: Journal of Chromatography B. - 1387-2273 .- 1878-5603. ; 722:1-2, s. 103-120
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Tidskriftsartikel (refereegranskat)abstract
- Size-exclusion chromatography has been used for fractionation of liposomes, proteoliposomes and biomembrane vesicles of up to approximately 500 nm in size and for separation of these entities from smaller components. Liposome sizes, encapsulation stability, and solute affinities for membrane proteins have been determined. Counter-current distribution in aqueous two-phase systems has widened the range of applications to larger structures. Immobilized biomembrane vesicles and (proteo)liposomes provide stationary phases for chromatographic analysis of specific or nonspecific membrane-solute interactions.
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