MECHANISMS AND REGULATION OF EPITHELIAL TRANSPORT

肾近端小管和肠刷状缘膜含有 催化Na+和葡萄糖的主动共转运的转运蛋白。 使用免疫学和功能标准，我们已经确定了一个 肾刷状缘膜中75 kD糖蛋白作为一个亚单位， Na+/葡萄糖同向转运体，纯化75 kD蛋白， 同质性和获得的功能重建， 蛋白脂质体。 我们的单克隆和多克隆抗体还 识别LLC-PK 1细胞中的75 kD亚基，LLC-PK 1细胞是来自 肾近曲小管 继续实施该项目的总体目标是 进一步表征肾脏Na+/D-的结构和功能， 葡萄糖同向转运体，并探讨其调控 在细胞培养中分化表达。 肾Na+/葡萄糖75 kD亚基cDNA的克隆 将对共转运体进行克隆和测序。 LLC-PK1 lambda gt11 筛选富含同向转运体cDNA的表达文库 使用我们的同向转运体的多克隆抗体;或者，LLC- PK 1 λ gt 10文库将使用合成的 对应于衍生的肽的序列的寡核苷酸 从我们的纯同向转运体制备。 预测的氨基酸 序列和二级结构将根据 cDNA序列。 同向转运体的寡聚体结构将通过 凝胶过滤和蔗糖梯度中的比较沉降 由D2 O和H2O以及通过交联研究制成。 的 Na+、葡萄糖或根皮苷影响寡聚体可能性 结构也将进行测试。 共转运蛋白的表位 膜的内外表面和跨膜段 将使用单克隆抗体对纯化的蛋白质进行定位 和定点多克隆抗体。 定点诱变 将开展研究，调查 同向转运体。 LLC-PK 1细胞Na+/葡萄糖转运体表达的调控 将使用特异性抗体进行研究，以监测 蛋白质和cDNA探针的生物合成和周转，以测量 特定的mRNA水平、转录速率和半衰期。 这些 研究将集中在同向转运体的戏剧性（20倍）诱导 用分化诱导剂处理后达到的活性 双乙酰胺（HMBA）。

Renal proximal tubule and intestinal brush border membranes contain transporters which catalyze active cotransport of Na+ and glucose. Using immunological and functional criteria, we have identified a 75 kD glycoprotein in renal brush border membranes as a subunit of the Na+/glucose symporter, purified the 75 kD protein to homogeneity and obtained functional reconstitution in proteoliposomes. Our monoclonal and polyclonal antibodies also recognize the 75 kD subunit in LLC-PK1 cells, a long term line from renal proximal tubule. The overall goals of the continuation of this project are to further characterize the structure and function of the renal Na+/D- glucose symporter and to investigate the regulation of its differentiated expression in cell culture. The cDNA encoding the 75 kD subunit of the renal Na+/glucose symporter will be cloned and sequenced. An LLC-PK1 lambda gt11 expression library enriched in symporter cDNA will be screened using our polyclonal antibody to the symporter; alternately, a LLC- PK1 lambda gtlO library will be screened using synthetic oligonucleotides corresponding to the sequence of a peptide derived from our pure symporter preparation. A predicted amino acid sequence and secondary structure will be calculated based on the cDNA sequence. The oligomeric structure of the symporter will be investigated by gel filtration and comparative sedimentation in sucrose gradients made from D2O and H2O and by cross-linking studies. The possibility that Na+, glucose or phlorizin affect oligomeric structure will also be tested. Epitopes of the symporter on the inner and outer surface of the membrane and transmembrane segment will be mapped using monoclonal antibodies to the purified protein and site-directed polyclonal antibodies. Site-directed mutagenesis studies will be initiated to investigate functional domains of the symporter. The regulation of Na+/glucose symporter expression in LLC-PK1 cells will be investigated using specific antibodies to monitor biosynthesis and turnover of the protein and cDNA probes to measure specific mRNA levels, rates of transcription and half-life. These studies will focus on the dramatic (20-fold) induction of symporter activity achieved after treatment with the differentiation inducer hexamethylene bisacetamide (HMBA).