GLUTATHIONE DEGRADING ENZYMES OF MICROVILLUS MEMBRANES

肾近端小管上皮细胞积极参与重吸收 大量的化合物和流体。这种传输发生在 特殊结构的单元格、笔刷边框，它由 面向肾小管腔的大量堆积的微绒毛。这个 上皮细胞产生和维持的生化过程 与基侧膜不同的微绒毛膜是 未知。这项研究计划的总体目标是研究 细胞达到并维持这一极性的机制。三 参与细胞表面降解的微绒毛酶 谷胱甘肽、谷氨酰转肽酶、氨基肽酶M和a 二肽酶，将作为模型系统来研究生物发生， 微绒毛蛋白的加工和细胞内的拓扑发生。这个 具体目标包括：1.结构和拓扑研究隔离， 单链前驱体的表征和加工 二聚酶，γ-谷氨酰转肽酶； 这些酶与膜的结合及其在体内的拓扑 这些膜。 2.微绒毛酶的拓扑发生--细胞内转运和 实现其最终目的地的分类机制；首字母 这三种酶的生物合成和加工过程中发生的事件。这个 操作方法包括经典的蛋白质和酶方法。 化学、免疫化学、免疫电子显微镜、脉冲追逐研究 肾组织切片，并对其体外翻译产物进行研究 肾脏基因的表达。

Renal proximal tubule epithelial cell is actively engaged in reabsorption of a large number of compounds and fluid. This transport occurs across a specialized structure of the cell, the brush border, which consists of numerous, packed microvilli facing the renal tubular lumen. The biochemical processes by which the epithelial cell generates and maintains the microvillus membranes distinct from the basal-lateral membranes is unknown. The overal objectives of this research proposal are to study mechanisms whereby the cell achieves and maintains this polarity. Three microvillus enzymes involved in the cell surface degradation of glutathione, gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase, will be used as model systems to study the biogenesis, processing, and intracellular topogenesis of microvillus proteins. The specific aims include: 1. Structural and Topological Studies isolation, characterization, and processing of the single-chain precursor of the dimeric enzyme, gamma-glutamyl transpeptidase; the structural basis for the association of these enzymes with the membranes and their topology within the membranes. 2. Topogenesis of the Microvillus Enzymes - intracellular transport and sorting mechanisms for achieving their final destination; the initial events in the biosynthesis and processing of the three enzymes. The methods of procedure include classical methods of protein and enzyme chemistry, immunochemistry, immunoelectron microscopy, pulse-chase studies with kidney slices, and studies on the in vitro translation products of the kidney mRNA.