May J M, Qu Z C, Beechem J M
Department of Medicine, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-2230.
Biochemistry. 1993 Sep 21;32(37):9524-31. doi: 10.1021/bi00088a002.
The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)
已证明人类红细胞葡萄糖转运蛋白的构象决定了其在一个大的细胞质环上对酶解的敏感性。我们采用相反的方法，研究了胰蛋白酶消化对该蛋白构象结构的影响。对去除蛋白质的红细胞膜进行彻底的胰蛋白酶消化，使残留转运蛋白对细胞松弛素B的亲和力降低了3倍，但不影响结合位点的总数。胰蛋白酶消化还增加了残留转运蛋白对D-葡萄糖和向内结合的糖苯基β-D-吡喃葡萄糖苷的亲和力，但降低了对向外结合的4,6-O-亚乙基葡萄糖的亲和力。这些结果表明，胰蛋白酶切割使剩余的转运蛋白稳定在内向构象中，但对细胞松弛素B的亲和力降低。纯化的重组葡萄糖转运蛋白的稳态荧光发射扫描不受胰蛋白酶消化的影响。添加浓度不断增加的碘化钾会产生线性的斯特恩-沃尔默图，该图也不受先前胰蛋白酶消化的影响。研究了色氨酸氧化剂N-溴代琥珀酰亚胺，以更灵敏地测量色氨酸环境。该试剂不可逆地抑制完整红细胞中的3-O-甲基葡萄糖转运和去除蛋白质的膜中细胞松弛素B的结合，每种活性在约0.3 - 0.4 nM时观察到半数最大效应。用N-溴代琥珀酰亚胺处理纯化的葡萄糖转运蛋白会导致色氨酸荧光随时间淬灭，通过全局分析的非线性回归将其分解为两个成分。胰蛋白酶消化减缓了反应较慢的一类色氨酸的氧化速率。
