Neeb M, Kunz U, Koepsell H
J Biol Chem. 1987 Aug 5;262(22):10718-27.
D-Glucose-binding polypeptides in the Na+-D-glucose cotransporter from pig renal cortex were identified by affinity labeling with two D-glucose analogs, 10-N-(N-[4-azido-2-nitrophenyl]-beta-alanyl)amino-1-decyl-beta-D- glucopyranoside (NapADG) and 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG). During short-term incubation in the dark, NapADG and BADG are reversible inhibitors of Na+ gradient-dependent D-glucose uptake and Na+-dependent phlorizin binding with Ki values of about 40 and 400 microM, respectively. Irreversible inhibition of Na+-dependent phlorizin binding, which was prevented by D-glucose or phlorizin, was measured after a 1-h incubation with BADG. Both NapADG and BADG selectively labeled polypeptides with apparent molecular weights of 82,000, 75,000, 64,000, and 47,000. Since labeling of the Mr 82,000 and 75,000 polypeptides by both analogs was partially dependent on the presence of Na+ and was partially protected by D-glucose or phlorizin but not by L-glucose or D-mannose, these polypeptides are thought to be components of the renal Na+-D-glucose cotransporter which contain D-glucose-binding sites. For the Mr 64,000 and 47,000 polypeptides, Na+ dependence and D-glucose protection were not constantly observed. However, also, these polypeptides are thought to be components or proteolytic splitting products of the Na+-D-glucose cotransporter since we observed that three monoclonal antibodies showed cross-reaction with the BADG-labeled Mr 82,000, 64,000, and 47,000 polypeptides (K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, and H. Koepsell, manuscript in preparation). When the BADG-labeled Mr 82,000 and 75,000 polypeptides were analyzed after two-dimensional separation by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three-labeled, D-glucose-protectable polypeptides with the respective molecular weights and isoelectric points of 82,000 and 5.6, 75,000 and 5.4, and 75,000 and 6.9 were distinguished. The data indicate that renal brush-border membranes contain several polypeptides which are components of the Na+-D-glucose cotransporter and contain D-glucose-binding sites.
通过用两种D - 葡萄糖类似物10 - N -（N - [4 - 叠氮基 - 2 - 硝基苯基] - β - 丙氨酰基）氨基 - 1 - 癸基 - β - D - 吡喃葡萄糖苷（NapADG）和10 - N -（溴乙酰基）氨基 - 1 - 癸基 - β - D - 吡喃葡萄糖苷（BADG）进行亲和标记，鉴定了猪肾皮质钠 - D - 葡萄糖共转运蛋白中的D - 葡萄糖结合多肽。在黑暗中短期孵育期间，NapADG和BADG是钠梯度依赖性D - 葡萄糖摄取和钠依赖性根皮苷结合的可逆抑制剂，其Ki值分别约为40和400μM。在用BADG孵育1小时后，测量了钠依赖性根皮苷结合的不可逆抑制，该抑制可被D - 葡萄糖或根皮苷阻止。NapADG和BADG均选择性地标记了表观分子量为82,000、75,000、64,000和47,000的多肽。由于两种类似物对82,000和75,000道尔顿多肽的标记部分依赖于钠的存在，并且部分受到D - 葡萄糖或根皮苷的保护，但不受L - 葡萄糖或D - 甘露糖的保护，因此这些多肽被认为是肾钠 - D - 葡萄糖共转运蛋白的组成部分，含有D - 葡萄糖结合位点。对于64,000和47,000道尔顿的多肽，并非始终观察到钠依赖性和D - 葡萄糖保护作用。然而，这些多肽也被认为是钠 - D - 葡萄糖共转运蛋白的组成部分或蛋白水解裂解产物，因为我们观察到三种单克隆抗体与BADG标记的82,000、64,000和47,000道尔顿多肽发生交叉反应（K. Korn，A. Raszeja - Specht，S. Bernotat - Danielowski和H. Koepsell，正在准备的手稿）。当通过等电聚焦和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳进行二维分离后分析BADG标记的82,000和75,000道尔顿多肽时，区分出三种标记的、D - 葡萄糖可保护的多肽，其分子量和等电点分别为82,000和5.6、75,000和5.4以及75,000和6.9。数据表明，肾刷状缘膜含有几种多肽，它们是钠 - D - 葡萄糖共转运蛋白的组成部分，并含有D - 葡萄糖结合位点。
