| 1. |
- Beigi, Farideh, et al.
(author)
-
Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport
- 1998
-
In: International Journal of Pharmaceutics. - 0378-5173 .- 1873-3476. ; 164:1-2, s. 129-137
-
Journal article (peer-reviewed)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.
|
|
| 2. |
|
|
| 3. |
- Gottschalk, Ingo, et al.
(author)
-
Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter
- 2000
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:23, s. 6875-6882
-
Journal article (peer-reviewed)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.
|
|
| 4. |
- Lundqvist, Andreas, et al.
(author)
-
Frontal affinity chromatographic analysis of membrane protein reconstitution
- 1997
-
In: Materials science & engineering. C, biomimetic materials, sensors and systems. - 0928-4931 .- 1873-0191. ; 4:4, s. 221-226
-
Journal article (peer-reviewed)abstract
- The human red cell glucose transporter Glut1 was solubilized with octaoxyethylene n-dodecyl ether (low critical micelle concentration (CMC)), purified, mixed with egg phospholipids and cholate, and reconstituted by gel filtration on Superdex 75. Free protepliposomes showed relatively high D-glucose transport activity. Frontal affinity chromatographic analysis with the proteoliposomes sterically immobilized in Superdex 200 gel beads revealed that the number of operative cytochalasin B (CB) binding sites increased during the first days of chromatographic runs to become the same as with 1-O-n-octyl β-d-glucopyranoside (high CMC) as solubilizer and Sephadex G-50 as gel filtration medium. The average number of sites per Glut1 monomer was 0.32 ± 0.02. The average Kd for CB was 66 ± 3 nM at 150 mM NaCl, similarly as for Glut1 in membrane vesicles, whereas the affinity of d-glucose for reconstituted Glut1 was lower (Kd = 44 ± 3 mM) than for membranous Glut1 (Kd = 15 ± 5 mM). Two theoretical treatments of affinity chromatographic data gave the same values in agreement with competitive and monovalent interactions.
|
|
