Peerce B E, Clarke R D
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
J Biol Chem. 1990 Jan 25;265(3):1731-6.
The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.
采用三步法从刷状缘膜囊泡中分离肠道Na⁺/葡萄糖共转运体，并以Na⁺依赖性根皮苷结合作为共转运体富集的指标。第一步是用0.02%的十二烷基硫酸钠（SDS）处理Ca²⁺沉淀的刷状缘膜囊泡，然后进行蔗糖梯度离心，这使得Na⁺/葡萄糖共转运体富集了5倍。第二步是在pH 7.4至pH 4.0的范围内进行色谱聚焦层析。与作为起始材料的SDS - 刷状缘膜囊泡蛋白相比，这一步导致了额外20倍的纯化。最后一步是在伴刀豆球蛋白A - 琼脂糖上进行亲和层析，这使得经色谱聚焦的蛋白富集了5倍。来自伴刀豆球蛋白A柱的糖蛋白部分重构成磷脂酰胆碱：胆固醇脂质体后，表现出Na⁺依赖性、根皮苷敏感性和渗透压敏感性葡萄糖摄取。在用银染色的SDS - 聚丙烯酰胺凝胶电泳上，该部分由一条单一的75 kDa多肽组成。基于这些标准，似乎已经开发出了一种分离Na⁺/葡萄糖共转运体的方案。
