Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-b inding site on every second GLUT1 monomer (state 1) and the other showing o ne site per monomer (state 2). Quantitative affinity chromatography of cyto chalasin B was performed on (a) biotinylated red blood cells, (b) cytoskele ton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposome s. The cells were adsorbed on streptavidin-derivatized gel beads, and the v esicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed b y 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 co nverted into state 1 on entrapment of the vesicles. Purification of GLUT1 i n the presence of non-ionic detergent followed by reconstitution produced G LUT1 in state 1. This state was maintained after entrapment of the proteoli posomes. 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) interact ion between the dextran-grafted agarose gel matrix and the membrane vesicle s, and (d) reconstitution to form proteoliposomes.