MECHANISMS AND REGULATION OF EPITHELIAL TRANSPORT

该项目的总体目标是识别和分离肾 探讨Na+/D-葡萄糖转运蛋白的调控机制 在已建立的肾上皮细胞系LLC-PK 1中表达 使用特异性抗血清的近端小管起源。 肾Na+/D-葡萄糖转运蛋白的抗体将由两个 不同的策略。 一种方法是基于形成 抗根皮苷抗体的抗独特型抗体 血清白蛋白结合物。 另一种方法是基于生产 用部分纯化的单克隆抗体免疫后 Na+/D-葡萄糖转运蛋白的制备。 免疫亲和方法将用于识别和分离肾 来自猪肾和来自LLC-PK 1细胞的Na+/D-葡萄糖转运蛋白 cultures. 分离的蛋白质将通过功能和生物学特性来表征。 分子标准 将进行交联研究， 研究其在细胞膜中可能的寡聚体结构。 Na+/D-葡萄糖的生物合成及其表达调控 将使用这些抗体研究LLC-PK 1培养物中的转运蛋白 probes. 生物合成标记和免疫沉淀研究，无细胞 翻译，免疫电印迹分析，交叉免疫电泳和 免疫荧光组织化学方法探讨其基础 对于显著增加的Na+/D-葡萄糖转运活性， 在细胞汇合后和分化增加后发展 诱导剂。

The overall goals of this project are to identify and isolate the renal Na+/D-glucose transporter(s) and to investigate the regulation of its expression in the established renal epithelial cell line LLC-PK1 of proximal tubule origin using specific antisera. Antibodies to the renal Na+/D-glucose transporter will be prepared by two different strategies. One approach is based on the formation of anti-idiotype antibodies prepared against antibodies to a phlorizin-bovine serum albumin conjugate. The other approach is based on production of monoclonal antibodies after immunization with partially - purified preparations of the Na+/D-glucose transporter. Immuno-affinity methods will be utilized to identify and isolate the renal Na+/D-glucose transporter(s) from pig kidney and from LLC-PK1 cell cultures. The isolated protein will be characterized by functional and molecular criteria. Cross-linking studies will be carried out to investigate its possible oligomeric structure in cell membranes. The biosynthesis and regulation of the expression of the Na+/D-glucose transporter in LLC-PK1 cultures will be investigated using these antibody probes. Biosynthetic labelling and immunoprecipitation studies, cell-free translation, immunoelectroblot analysis, crossed immunoelectrophoresis and immunofluorescense histochemistry will be carried out to explore the basis for the dramatically-increased Na+/D-glucose transport activity which develops after cell confluence and after addition of differentiation inducers.