Janoshazi A, Kifor G, Solomon A K
Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 02115.
J Membr Biol. 1991 Sep;123(3):191-207. doi: 10.1007/BF01870403.
We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.
我们之前已经表明，人类红细胞葡萄糖转运蛋白和阴离子交换蛋白（带3蛋白）紧密接触，以至于信息可以从葡萄糖转运蛋白传递到带3蛋白。本实验旨在表明信息是否可以反向传递，利用色氨酸荧光的变化来报告葡萄糖转运蛋白的构象。为了确定色氨酸荧光变化是否可归因于葡萄糖转运蛋白，我们的实验基于Helgerson和Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464 - 5475] 所采用的程序，将细胞松弛素B（CB），即特异性D - 葡萄糖转运抑制剂，从其在葡萄糖转运蛋白内表面的结合位点上置换出来，并且我们表明这些程序改变了色氨酸荧光。添加75 mM麦芽糖（一种不可转运的二糖，它也能置换CB）会使新鲜红细胞中的色氨酸荧光随时间呈双相增强，这种增强受到特异性阴离子交换抑制剂DBDS（4,4'-二苯甲酰胺基 - 2,2'-二苯乙烯二磺酸盐）的调节。在对另外九种二糖的研究中，我们发现双相动力学和DBDS效应都取决于特定的二糖构象，这表明这两种效应可归因于一个对糖构象敏感的位点。长期（800秒）实验表明，麦芽糖结合（±DBDS）会导致在整个800秒观察期内持续的阻尼非谐振荡。对这些振荡的温度依赖性进行数学分析表明，2 microM DBDS将阻尼项活化能从9.5±2.8 kcal mol⁻¹ deg⁻¹提高了四倍，达到39.7±5.1 kcal mol⁻¹ deg⁻¹，这为葡萄糖转运蛋白和带3蛋白之间的信号双向传递这一观点提供了有力支持。
