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-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.
Gottschalk
I
I
Department of Biochemistry, Biomedical Center, Uppsala University, Sweden.
Lundqvist
A
A
Zeng
C M
CM
Hägglund
C L
CL
Zuo
S S
SS
Brekkan
E
E
Eaker
D
D
Lundahl
P
P
D001665:Binding Sites; D001692:Biological Transport; D019921:Biotinylation; D002846:Chromatography, Affinity; D003571:Cytochalasin B / Q000737:chemistry*; D003599:Cytoskeleton / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D051272:Glucose Transporter Type 1; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C495677:SLC2A1 protein, human; C020485:proteoliposomes; D003571:Cytochalasin B; D012685:Sepharose