PROTEIN TRANSLOCATION AND PROCESSING IN HEN OVIDUCT

拟议的项目将研究母鸡输卵管信号肽酶（SP）， 一种膜结合蛋白水解酶， 来自新生分泌蛋白的信号肽， 运输到内质网的内腔。 纯化 输卵管SP由两种蛋白质组成：19 kDa多肽和 一种疏水异质的23 kDa糖肽。 氨基 胰蛋白酶肽的酸序列分析 不同的糖蛋白表明它们是 相同多肽的差异糖基化形式。 的 研究将确定完整的一级结构， 输卵管SP使用直接氨基酸序列的组合 纯化的肽的分析和SP cDNA的分子克隆。 这些研究将详细描述主要的 真核生物信号肽酶的结构， 是成功生物合成大多数分泌的 proteins. 拟议中的研究将审查 输卵管SP的两种蛋白质，以确定其性质 关联，并确定是否两者都需要酶 活动 将尝试分离这两种蛋白质 而不损失酶活性， 确定了酶与变性剂的浓度， 酶活性 光交联实验，使用 光活化的合成信号肽类似物将用于 鉴定与信号相互作用的输卵管SP蛋白 肽。 实验工作将集中在表征 利用合成信号研究输卵管菌酶学 肽类似物和突变信号肽将鉴定 信号肽切割的结构要求。 活性部位- 将鉴定输卵管SP催化的定向抑制剂。 不可逆抑制剂的鉴定将导致 酶的活性位点的鉴定和产率 关于催化机制的信息。 成功完成 建议的研究将带来详细的了解， 这把钥匙的一级结构和基本生物化学 细胞生物合成途径中的酶。

The proposed project will study hen oviduct signal peptidase (SP), a membrane-bound proteolytic enzyme that removes amino terminal signal peptides from nascent secretory proteins as they are transported into the lumen of the endoplasmic reticulum. Purified oviduct SP is composed of two proteins: a 19 kDa polypeptide and an electrophoretically heterogenous 23 kDa glycopeptide. Amino acid sequence analysis of tryptic peptides derived from the electrophoretically distinct glycoproteins have shown them to be differentially glycosylated forms of the same polypeptide. The proposed study will determine the complete primary structure of oviduct SP using a combination of direct amino acid sequence analysis of purified peptides and by molecular cloning of SP cDNAs. These studies will provide a detailed description of the primary structure of a eukaryotic signal peptidase, one of the components absolutely required for successful biosynthesis of most secreted proteins. The proposed studies will examine the association of the two proteins of oviduct SP to determine the nature of their association and to identify whether both are required for enzymatic activity. Attempts will be made to dissociate the two proteins without loss of enzymatic activity by treating the active, purified enzyme with concentrations of denaturants determined to preserve enzymatic activity. Photocrosslinking experiments using photoactivated synthetic signal peptide analogs will be used to identify the protein(s) of oviduct SP that interact with the signal peptide. Experimental efforts will focus on characterization of the enzymology of oviduct SP. Studies with synthetic signal peptide analogs and mutant signal peptides will identify the structural requirements for signal peptide cleavage. Active site- directed inhibitors of oviduct SP catalysis will be identified. Identification of an irreversible inhibitor will lead to the identification of the active site of the enzyme and yield information about the catalytic mechanism. Successful completion of the proposed studies will bring about a detailed understanding of the primary structure and fundamental biochemistry of this key enzyme in the biosynthetic pathway of cells.