Martin G E, Seamon K B, Brown F M, Shanahan M F, Roberts P E, Henderson P J
Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
J Biol Chem. 1994 Oct 7;269(40):24870-7.
Forskolin is a potent inhibitor of mammalian passive glucose transporters. Here we show that forskolin is a remarkably specific inhibitor of energized D-galactose transport by the GalP sugar-H+ symport protein of Escherichia coli. Surprisingly, it does not inhibit transport of L-arabinose or D-xylose by the related E. coli AraE and XylE transporters, even though the amino acid sequences of their proteins are 30-64% identical to GalP and to the mammalian GLUT family. However, unlike GLUT1, photoactivation of the [3H]forskolin-GalP complex fails to incorporate radioactivity covalently into the protein, in contrast to the effective incorporation of radioactivity from [3H]cytochalasin B into both proteins. However, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskol in ([125I]APS-forskolin), which labels GLUT1, is a potent labeling reagent for GalP and, to a lesser extent, for AraE. The appropriate sugar substrates of each transporter protect it against the [125I]APS-forskolin. Equilibrium binding studies using membranes from an E. coli strain that overexpresses GalP reveal a single set of high affinity binding sites for [3H]forskolin with a Kd of 1.3-1.4 microM, probably forming a 1:1 complex, compared with a value of 7.5 microM for GLUT1. Sugar substrates of GalP and cytochalasin B displace forskolin from the protein. The nonhomologous sugar-H+ symporters for L-rhamnose (RhaT), L-fucose (FucP) and lactose (LacY) in E. coli are insensitive to forskolin. Forskolin and [125I]APS-forskolin, therefore, constitute novel probes for exploring the structure-activity relationship of the bacterial GalP protein. GalP will provide an excellent model for the human glucose transporters and for elucidating the molecular basis of subtle differences in substrate and inhibitor recognition by individual members of this widespread family of transport proteins.
福斯高林是哺乳动物被动葡萄糖转运蛋白的强效抑制剂。在此我们表明，福斯高林是大肠杆菌GalP糖-H⁺同向转运蛋白介导的有能量的D-半乳糖转运的显著特异性抑制剂。令人惊讶的是，它并不抑制相关的大肠杆菌AraE和XylE转运蛋白对L-阿拉伯糖或D-木糖的转运，尽管它们蛋白质的氨基酸序列与GalP以及哺乳动物GLUT家族的氨基酸序列有30 - 64%的同一性。然而，与GLUT1不同，[³H]福斯高林-GalP复合物的光活化未能将放射性共价掺入蛋白质中，与之形成对比的是，[³H]细胞松弛素B的放射性有效地掺入了这两种蛋白质中。然而，标记GLUT1的3-[¹²⁵I]碘-4-叠氮苯乙酰胺基-7-O-琥珀酰去乙酰基福斯高林（[¹²⁵I]APS-福斯高林）是GalP的强效标记试剂，对AraE的标记作用较弱。每种转运蛋白的合适糖底物可保护其免受[¹²⁵I]APS-福斯高林的作用。使用过表达GalP的大肠杆菌菌株的膜进行的平衡结合研究显示，[³H]福斯高林有一组单一的高亲和力结合位点，Kd为1.3 - 1.4微摩尔，可能形成1:1复合物，而GLUT1的Kd值为7.5微摩尔。GalP的糖底物和细胞松弛素B可将福斯高林从蛋白质上置换下来。大肠杆菌中L-鼠李糖（RhaT）、L-岩藻糖（FucP）和乳糖（LacY）的非同源糖-H⁺同向转运蛋白对福斯高林不敏感。因此，福斯高林和[¹²⁵I]APS-福斯高林构成了探索细菌GalP蛋白结构-活性关系的新型探针。GalP将为人类葡萄糖转运蛋白以及阐明这个广泛的转运蛋白家族中各个成员在底物和抑制剂识别方面细微差异的分子基础提供一个极好的模型。
