Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.
Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.
复制标题
GLUT1糖进出口位点之间合作相互作用的停流分析。
DOI：
10.1021/bi990130o
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Carruthers,A
中科院分区：
文献类型：
--
作者：
Sultzman,LA;Carruthers,A
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 increasek-1(app)and reducek1(app)for cytochalasin B interaction with GLUT1. Higher sugar concentrations decreasek1(app)further. The simple carrier mechanism predicts thatk1(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.
