Chromatographic characterization of the human red cell glucose transporter Glut1 in the cells, in membrane vesicles and in proteoliposomes

Abstract: The human glucose transporter Glut1 is used by many cells, especially red blood cells andbrain cell, to take up glucose and other necessary metabolites. Gentle chromatographic methods can be applied for analyses of Glut1 activities in lipid environments similar to the native one. For this purpose, methods for immobilization of cells, cytoskeleton-depleted red cell membrane vesicles and proteoliposomes in chromatographic gel media have been developed and refined. The vesicles and proteoliposomes were immobilized by freeze-thaw-induced fusion in dextran-grafted agarose gel beads (Superdex 200). The sheltered environment in microcavities kept the Glut1 active for repeated analyses over time periods up to three months at room temperature.Frontal affinity chromatography, Hummel and Dreyer analyses and centrifugation methods revealed that the Glut1 affinities for inhibitors and D-glucose were highest in the cells andbecame successively lower upon cytoskeleton-depletion and subsequent solubilization andreconstitution. The affinity for D-glucose was higher at 37°C than at lower temperatures and varied with the pH. The cytochalasin-B binding characteristics of Glut1 changed upon reconstitution and, in the case of Glut1 in membrane vesicles, also upon immobilization. Glut1 in free membrane vesicles bound one cytochalasin-B molecule per monomer at saturation, whereas this ratio was halved upon immobilization of the vesicles to become equal to the ratio for Glut1 in proteoliposomes.