MECHANISMS AND REGULATION OF EPITHELIAL TRANSPORT

点击翻译按钮获取中文摘要

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.