Chen S R, Lo T C
Department of Biochemistry, University of Western Ontario, London, Canada.
Biochem J. 1988 Apr 1;251(1):63-72. doi: 10.1042/bj2510063.
We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.
我们最近证明，未分化的大鼠L6成肌细胞中存在两种己糖转运系统：D-葡萄糖和2-脱氧-D-葡萄糖优先通过高亲和力系统转运，而3-O-甲基-D-葡萄糖主要通过低亲和力系统转运。发现突变体D23仅在高亲和力己糖转运系统中存在缺陷。低亲和力转运系统对细胞松弛素B（CB）的抑制作用更为敏感。本研究通过测量CB与全细胞和质膜的结合，研究了CB结合位点的特性、性质和调节。对结合数据的Scatchard分析揭示了存在两个CB结合位点，即CBH和CBL。这两个位点不仅对CB的亲和力不同，而且它们的水平也可以通过各种生化、生理和基因操作而发生不同的改变。CBL类似于高亲和力己糖转运系统，因为它在突变体D23中不存在，并且在葡萄糖饥饿的细胞中大量存在。此外，CB与该位点的结合受到D-葡萄糖和2-脱氧-D-葡萄糖的抑制，它们是高亲和力己糖转运系统的优选底物。另一方面，发现CBH在突变体D23中未改变，该突变体也保留了正常的低亲和力己糖转运系统。CBH也类似于低亲和力转运系统，因为它在葡萄糖饥饿的细胞中没有升高。此外，CB与该位点的结合可以被3-O-甲基-D-葡萄糖抑制，它是低亲和力转运系统的优选底物。应该注意的是，2-脱氧-D-葡萄糖对CBH没有太大影响，反之亦然。对纯化膜制剂的研究表明，两个CB结合位点在质膜和低密度微粒体部分中的比例相似。质膜研究还表明，6-磷酸-D-葡萄糖而不是6-磷酸-2-脱氧-D-葡萄糖在激活CB结合方面非常有效。所提供的数据表明，CB结合可能以变构方式受到糖类似物的调节。
