Cloherty E K, Heard K S, Carruthers A
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605, USA.
Biochemistry. 1996 Aug 13;35(32):10411-21. doi: 10.1021/bi953077m.
GLUT1-mediated, passive D-glucose transport in human erythrocytes is asymmetric, Vmax and K(m)(app) for D-glucose uptake at 4 degrees C are 10-fold lower than Vmax and K(m)(app) for D-glucose export. Transport asymmetry is not observed for GLUT1-mediated 3-O-methylglucose transport in rat, rabbit, and avian erythrocytes and rat adipocytes where Vmax for sugar uptake and exit are identical. This suggests that transport asymmetry is either an intrinsic catalytic property of human GLUT1 or that factors present in human erythrocytes affect GLUT1-mediated sugar transport. In the present study we assess human erythrocyte sugar transport asymmetry by direct measurement of sugar transport rates and by analysis of the effects of intra- and extracellular sugars on cytochalasin B binding to the sugar export site. We also perform internal consistency tests to determine whether the measured, steady-state 3-O-methylglucose transport properties of human erythrocytes agree with those expected of two hypothetical models for protein-mediated sugar transport. The simple-carrier hypothesis describes a transporter that alternately exposes sugar import and sugar export pathways. The fixed-site carrier hypothesis describes a sugar transporter that simultaneously exposes sugar import and sugar export pathways. Steady-state 3-O-methylglucose transport in human erythrocytes at 4 degrees C is asymmetric. Vmax and K(m)(app) for sugar uptake are 10-fold lower than Vmax and K(m)(app) for sugar export. Phloretin-inhibitable cytochalasin B binding to intact red cells is unaffected by extracellular D-glucose but is competitively inhibited by intracellular D-glucose. This inhibition is reduced by 13% +/- 4% when saturating extracellular D-glucose levels are also present. Assuming transport is mediated by a simple-carrier and that cytochalasin B and intracellular D-glucose binding sites are mutually exclusive, the cytochalasin B binding data are explained only if transport is almost symmetric (Vmax exit = 1.4 Vmax entry). The cytochalasin B binding data are consistent with both symmetric and asymmetric fixed-site carriers. Analysis of 3-O-methylglucose, 2-deoxy-D-glucose, and D-glucose uptake in the presence of intracellular 3-O-methylglucose, demonstrates significant divergence in experimental and theoretical transport behaviors. We conclude either that human erythrocyte sugar transport is mediated by a carrier mechanism that is fundamentally different from those considered previously or that human erythrocyte-specific factors prevent accurate determination of GLUT1-mediated sugar translocation across the cell membrane. We suggest that GLUT1-mediated sugar transport in all cells is an intrinsically symmetric process but that intracellular sugar complexation in human red cells prevents accurate determination of transport rates.
人类红细胞中由葡萄糖转运蛋白1（GLUT1）介导的被动D - 葡萄糖转运是不对称的，4℃时D - 葡萄糖摄取的最大转运速率（Vmax）和表观米氏常数（K(m)(app)）比D - 葡萄糖输出的Vmax和K(m)(app)低10倍。在大鼠、兔子和鸟类红细胞以及大鼠脂肪细胞中，GLUT1介导的3 - O - 甲基葡萄糖转运未观察到转运不对称性，其中糖摄取和输出的Vmax是相同的。这表明转运不对称性要么是人类GLUT1的固有催化特性，要么是人类红细胞中存在的因素影响了GLUT1介导的糖转运。在本研究中，我们通过直接测量糖转运速率以及分析细胞内和细胞外糖类对细胞松弛素B与糖输出位点结合的影响，来评估人类红细胞糖转运的不对称性。我们还进行了内部一致性测试，以确定所测量的人类红细胞稳态3 - O - 甲基葡萄糖转运特性是否与蛋白质介导的糖转运的两个假设模型所预期的特性一致。简单载体假说描述了一种交替暴露糖导入和糖输出途径的转运体。固定位点载体假说描述了一种同时暴露糖导入和糖输出途径的糖转运体。4℃时人类红细胞中稳态3 - O - 甲基葡萄糖转运是不对称的。糖摄取的Vmax和K(m)(app)比糖输出的Vmax和K(m)(app)低10倍。根皮素抑制的细胞松弛素B与完整红细胞的结合不受细胞外D - 葡萄糖的影响，但受到细胞内D - 葡萄糖的竞争性抑制。当也存在饱和的细胞外D - 葡萄糖水平时，这种抑制作用降低了13%±4%。假设转运由简单载体介导，并且细胞松弛素B和细胞内D - 葡萄糖结合位点相互排斥，那么只有当转运几乎对称（Vmax输出 = 1.4 Vmax输入）时，细胞松弛素B结合数据才能得到解释。细胞松弛素B结合数据与对称和不对称的固定位点载体均一致。在细胞内存在3 - O - 甲基葡萄糖的情况下，对3 - O - 甲基葡萄糖、2 - 脱氧 - D - 葡萄糖和D - 葡萄糖摄取的分析表明，实验和理论转运行为存在显著差异。我们得出结论，要么人类红细胞糖转运由一种与先前考虑的机制根本不同的载体机制介导，要么人类红细胞特异性因素阻止了对GLUT1介导的糖跨细胞膜转运的准确测定。我们认为，所有细胞中GLUT1介导的糖转运本质上是一个对称过程，但人类红细胞中的细胞内糖络合作用阻止了对转运速率的准确测定。
