Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.
The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.
Sultzman
L A
LA
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655, USA.
Carruthers
A
A
D019325:3-O-Methylglucose / Q000097:blood / Q000737:chemistry; D001665:Binding Sites; D001692:Biological Transport; D001786:Blood Glucose / Q000737:chemistry / Q000378:metabolism; D003571:Cytochalasin B / Q000097:blood; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D051272:Glucose Transporter Type 1; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008081:Liposomes / Q000097:blood / Q000737:chemistry; D008320:Maltose / Q000097:blood / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000737:chemistry* / Q000502:physiology; D011510:Proteolipids / Q000097:blood / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D001786:Blood Glucose; D051272:Glucose Transporter Type 1; D008024:Ligands; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C495677:SLC2A1 protein, human; C020485:proteoliposomes; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D008320:Maltose