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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.

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.

Development of methods for rendering chromatography more applicable to studies in lifesciences remains an area of active research. In the present work, new methods inbiomembrane chromatography and microchromatographic techniques have been developedand applied successfully to quantitative analysis of biological interactions and separations ofproteins on a micro-scale. Biomembranes, specifically: human red cells/ghosts, membrane vesicles and proteoliposomes, were immobilized in gel beds for chromatographic analyses of solutes specificallybound to the glucose transporter Glut1 and the nucleoside transporter of the red cellmembrane. The resultant binding parameters revealed that this system can be a practical toolfor quantitative analysis of biological interactions involving integral membrane proteins andfor monitoring procedures for purification and reconstitution of membrane proteins. Analternative method based on Hummel and Dreyer analysis was introduced and proved usefulin analyses of cytochalasin-B binding to Glut1 in membrane vesicles and proteoliposomes. Chromatographic analysis of non-specific partitioning of a drug into lipid bilayers wasperformed on a microcolumn containing liposomes immobilized in the gel bed. The resultsshowed that this method can be utilized for predicting absorption of a drug in vivo.Hydrophobic interaction microchromatography of proteins and a blotting membranetechnique for detection of minute amounts of proteins have been developed on the basis ofcontinuous beds which can be synthesized directly in the microcolumn tubes. Thesedevelopments extend the uses of continuous beds to a broader field in life science.

The affinities of D-glucose and the transport inhibitors, forskolin and cytochalasin B (CB), for Glut1 were studied by frontal affinity chromatography at pH 5-10 on sterically immobilized proteoliposomes with reconstituted human red cell glucose transporter Glut1. The affinity of D-glucose for Glut1 became slightly weaker as the pH was increased. The inhibitor affinities decreased and became immeasurably weak above pH 9. At pH 7.4, the dissociation constants were 44 mM for glucose, 1.8 microM for forskolin and 72 nM for CB. The affinities of these solutes for Glut1 in red cell membrane vesicles and particularly for Glut1 in red cells were higher, as shown by chromatographic analyses.