| 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. |
- Boija, Elisabet, 1977-
(författare)
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Partitioning of Drugs and Lignin Precursor Models into Artificial Membranes
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall. In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models. Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models. Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.
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| 3. |
- 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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| 4. |
- Lagerquist Hägglund, Christine, 1973-
(författare)
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Affinity-, Partition- and Permeability Properties of the Human Red Blood Cell Membrane and Biomembrane Models, with Emphasis on the GLUT1 Glucose Transporter
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Lagerquist Hägglund, Christine, et al.
(författare)
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Centrifugal and chromatographic analyses of tryptophan and tyrosine uptake by red blood cells and GLUT1 proteoliposomes with permeability estimates and observations on dihydrocytochalasin B
- 2003
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 55:2, s. 127-140
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed transport into liposomes and proteoliposomes, separated the free and internalized radioactively labeled substrates by size-exclusion chromatography (SEC) and observed a net influx owing to nonfacilitated diffusion across the lipid bilayers during the separation. The permeabilities (10(-9) cm/s) of glucose transporter (GLUT1) proteoliposomes were estimated to be 4.6, 1.0, 1.4 and 2.1 for D-glucose, L-glucose, L-Tyr and L-Trp, respectively; 15, 3.3, 5.1 and 2.1 times higher than the corresponding permeabilities of liposomes. These values indicated that GLUT1 did not transport Tyr or Trp, or transported Tyr, and only Tyr, slowly. This interpretation was supported by further analyses. Dihydrocytochalasin B inhibited the transport of Tyr and, partially, Trp into human red blood cells (centrifugal analyses). It did not inhibit Tyr and Trp influx into GLUT1 proteoliposomes, but partitioned strongly into the bilayers and seemed to make them fragile. The GLUT1 inhibitor cytochalasin B and the GLUT1 substrate 2-deoxy-D-glucose did not inhibit Tyr transport into the cells. Upon immobilized biomembrane affinity chromatography, Trp decreased the cytochalasin B retardation by GLUT1 only at levels far above the physiological Trp concentration. Ethanol (commonly added to aqueous solutions for enhancing a compound's solubility) halved the retardation at 4% (v/v) concentration. Drastic modification of the SEC method is required to allow permeability measurements with nonlabeled and highly permeable substrates.
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| 6. |
- 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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| 7. |
- 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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| 8. |
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| 9. |
- 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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| 10. |
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