Cloherty E K, Sultzman L A, Zottola R J, Carruthers A
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605, USA.
Biochemistry. 1995 Nov 28;34(47):15395-406. doi: 10.1021/bi00047a002.
Human erythrocyte net sugar transport is hypothesized to be rate-limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules [Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249]. The present study examines these hypotheses. Protein-mediated 3-O-methylglucose uptake at 4 degrees C by human erythrocytes and by resealed, hypotonically lysed erythrocytes (ghosts) is inhibited by increasing solvent viscosity. Protein-mediated transport and transbilayer diffusion of the slowly transported substrate 6-NBD glucosamine are unaffected by increasing solvent viscosity. These findings suggest that protein-mediated 3-O-methylglucose transport is diffusion-limited in erythrocytes. More detailed analyses of red cell 3-O-methylglucose uptake (at 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling of a small compartment (C1 = 29 +/- 6% total cell volume; k1 = 7.4 +/- 1.7 min-1) and slow filling of a larger compartment (C2 = 71 +/- 6% total cell volume k2 = 0.56 +/- 0.11 min-1). Erythrocyte D-glucose net uptake at 4 degrees C is also a biphasic process. Transmembrane sugar leakage is a monoexponential process indicating that multicomponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limiting 3-O-methylglucose concentrations is described by single exponential kinetics. This demonstrates that multicomponent sugar uptake does not result from influx into two populations of cells with widely different sugar transporter content. We conclude that biexponential sugar uptake results from slow (relative to transport) exchange of sugars between serial, intracellular sugar compartments. Biexponential sugar uptake is observed under equilibrium exchange conditions (intracellular sugar concentration = extracellular sugar concentration) but only at 3-O-methylglucose concentrations of less than 1 mM. Above this sugar concentration, exchange uptake is a monoexponential process. Because diffusion rates are independent of diffusant concentration, this suggests that multicomponent uptake results from high-affinity sugar binding within the cell. The concentration of cytosolic binding sites (30 microM, Kd(app) = 400 microM) was estimated from the equilibrium cellular 3-O-methylglucose space. Biexponential net 3-O-methylglucose uptake is also observed in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure to hemin. This demonstrates that neither membrane-bound nor free cytosolic hemoglobin forms the sugar-binding complex.(ABSTRACT TRUNCATED AT 400 WORDS)
据推测，人类红细胞的净糖转运受限于糖在细胞溶质中的扩散减少和/或糖与细胞内大分子的可逆结合[Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235 - 249]。本研究对这些假设进行了检验。在4℃时，人红细胞以及经重封、低渗裂解的红细胞（血影）对蛋白质介导的3 - O - 甲基葡萄糖摄取会因溶剂黏度增加而受到抑制。缓慢转运的底物6 - NBD - 葡糖胺的蛋白质介导转运和跨膜扩散不受溶剂黏度增加的影响。这些发现表明，蛋白质介导的3 - O - 甲基葡萄糖转运在红细胞中受扩散限制。对红细胞摄取3 - O - 甲基葡萄糖（在4℃和细胞外糖水平受限的情况下）进行更详细的分析发现，净内流是一个双指数过程，其特征为一个小隔室的快速填充（C1 = 29±6%总细胞体积；k1 = 7.4±1.7分钟-1）和一个较大隔室的缓慢填充（C2 = 71±6%总细胞体积；k2 = 0.56±0.11分钟-1）。4℃时红细胞对D - 葡萄糖的净摄取也是一个双相过程。跨膜糖泄漏是一个单指数过程，这表明多组分、蛋白质介导的摄取并非源于不同细胞体积的两个细胞群体对糖的摄取。在3 - O - 甲基葡萄糖浓度受限的情况下，糖的外流由单指数动力学描述。这表明多组分糖摄取并非源于流入糖转运体含量差异很大的两个细胞群体。我们得出结论，双指数糖摄取是由于糖在连续的细胞内糖隔室之间进行缓慢（相对于转运）交换所致。在平衡交换条件下（细胞内糖浓度 = 细胞外糖浓度）观察到双指数糖摄取，但仅在3 - O - 甲基葡萄糖浓度低于1 mM时出现。高于此糖浓度时，交换摄取是一个单指数过程。由于扩散速率与扩散剂浓度无关，这表明多组分摄取是由细胞内高亲和力的糖结合引起的。根据细胞内3 - O - 甲基葡萄糖空间的平衡状态估算出细胞溶质结合位点的浓度（30μM，Kd(app)=400μM）。在人红细胞血影、对照人K562细胞以及通过长期暴露于血红素诱导合成血红蛋白的K562细胞中也观察到了双指数净3 - O - 甲基葡萄糖摄取。这表明膜结合血红蛋白和游离细胞溶质血红蛋白均未形成糖结合复合物。（摘要截选至400字）
