BIOSYNTHESIS OF RENAL GAMMA-GLUTAMYLTRANSPEPTIDASE

肾脏的近端小管细胞，负责 溶质和流体的矢量传输，其特征在于不对称的 质膜，使细胞具有不同的极性。 的机制 细胞获得并保持基底侧质膜 与顶端管腔刷状缘膜不同的是未知的。 最近 在类似的细胞类型（肠绒毛细胞）中的实验表明， 糖蛋白直接插入到两种膜中，但有些糖蛋白插入到两种膜中， 基底侧膜通过微管依赖性过程迁移到 刷状缘膜。 这项建议的目的是研究 肾刷状缘膜生物发生利用 γ-谷氨酰转肽酶作为特异性探针。 这片灌木丛边缘 膜水解酶被合成为单一糖蛋白（前肽）， 其大部分被共裂解或后裂解， 异二聚体结构。 较大的亚基含有膜锚 而较小的亚基包含活性位点的一部分。 的 残余的未裂解的前肽显然被加工并插入到 刷状缘膜，其动力学类似于异二聚体结构 在它被第二次蛋白水解反应裂解之前。 具体 该建议的目的包括：1）确定亚细胞途径 转肽酶插入培养的 肾细胞; 2）部分表征的寡糖部分， 纯化的转肽酶及其动力学的测定 体内加工; 3）鉴定的主要翻译产物的 转肽酶和不完全异源二聚体产生的基础， 内质网;和4）确定的命运， 转肽酶前肽在刷状缘膜。

The proximal tubule cell of the kidney, which is responsible for the vectorial transport of solutes and fluid, is characterized by an asymmetric plasma membrane which gives the cell distinct polarity. The mechanism by which the cell achieves and maintains a basal lateral plasma membrane distinct from an apical lumenal brush border membrane is unknown. Recent experiments in an analogous cell type (intestinal villus cells) suggest that glycoproteins are inserted directly into both membranes, but some in the basal lateral membrane migrate by a microtubule-dependent process into the brush border membrane. The objective of this proposal is to study renal brush border membrane biogenesis utilizing Gamma-glutamyltranspeptidase as a specific probe. This brush border membrane hydrolase is synthesized as a single glycoprotein (propeptide) and the majority of it is cleaved either co- or posttranslationally to yield a heterodimer structure. The larger subunit contains the membrane anchor while the smaller subunit contains a portion of the active site. The residual uncleaved propeptide is apparently processed and inserted into the brush border membrane with kinetics similar to the heterodimer structure before it is cleaved by a secondary proteolytic reaction. The specific aims of this proposal include: 1) determination of the subcellular route of insertion of transpeptidase into the brush border membrane of cultured renal cells; 2) partial characterization of the oligosaccharide moieties on purified transpeptidase and determination of the kinetics of their processing in vivo; 3) identification of the primary translation product of the transpeptidase and the basis for incomplete heterodimer production in the endoplasmic reticulum; and 4) determination of the fate of transpeptidase propeptide in the brush border membrane.