IN VITRO BIOSYNTHESIS OF INSULIN AND GLUCAGON

胰岛激素生长抑素(SRIF)、胰高血糖素和胰岛素 作为更大的前体合成的。我们的长期目标是确定 前体在调节细胞内运输中的作用， 翻译后加工和激素的分泌。PreproSRIF为 一个对这些研究有用的模型，因为它是最简单的多肽之一 激素前体，只包含一个生物活性多肽和一个 蛋白质分解加工部位，用于切除成熟激素。vbl.使用 重组DNA技术一系列突变的前ProSRIF分子将被 构建的目的是研究前体中的功能结构域。 定点突变将被用来改变体内的个别氨基酸。 蛋白水解区；缺失突变体缺乏确定的区域 的前肽将被构建并且该前肽将与 非分泌型蛋白质。这些cDNA将被导入到异源的 对细胞和分泌参数进行了考察。常见的排序和 酵母前原α因子和前原SRIF的处理结构域将是 通过表达天然前SRIF-cDNA和嵌合cDNA进行鉴定 在酵母中编码不同数量的这些前体。Cdna 编码前胰高血糖素原将被引入不同类型的细胞和 对它们的翻译后加工和分泌进行了研究。在……里面 与前胰高血糖素原相比，前胰高血糖素原编码几种与胰高血糖素相关的基因 多肽，因此代表前体的下一级复杂性 正在处理。翻译终止密码子将被引入胰岛素原 将在转录-翻译耦合系统中用于 产生截短的前胰岛素原。这些将被用来调查 可转移的新生多肽链的最小尺寸 内质网细胞膜。激素前体处理酶将使用一种 分子方法：从细胞中获得高水平的cDNA库 加工活性将被插入逆转录病毒表达载体 和重组病毒，用于感染具有SRIF前c DNA的L细胞 已经稳定整合，并且只分泌proSRIF，因为它们缺乏 激素前体的处理活动。成熟的SRIF的分泌将被检测 使用抗体免疫印迹程序；DNA将从 并鉴定为阳性细胞。确定结构性决定因素， 胰岛激素前体的中介加工与 了解它们的合成和分泌具有重要的意义 关于糖尿病的病因学。

The pancreatic islet hormones somatostatin (SRIF), glucagon and insulin are synthesized as larger precursors. Our long term goal is to determine the role of the presursors in mediating intracellular transport, post-translational processing and secretion of the hormones. PreproSRIF is a useful model for these studies since it is one of the simplest peptide hormone precursors, containing only a single bioactive peptide and one proteolytic processing site for excision of the mature hormone. Using recombinant DNA techniques a series of mutated preproSRIF molecules will be constructed to investigate functional domains in the precursor. Site-directed mutagenesis will be used to alter individual amino acids in the proteolytic processing site; deletion mutants lacking defined regions of the propeptide will be constructed and the propeptide will be fused with non-secretory proteins. These cDNAs will be transfected into heterologous cells and the parameters of secretion investigated. Common sorting and processing domains in yeast preproalpha factor and preproSRIF will be identified by expressing native prepro-SRIF cDNAs and chimeric cDNAs encoding variable amounts of these respective precursors in yeast. cDNA encoding preproglucagons will be introduced into different cell types and their post-translational processing and secretion investigated. In contrast to preproSRIF, preproglucagons encode several glucagon-related peptides and thus represent the next order of complexity of precursor processing. Translation stop codons will be introduced into preproinsulin cDNA which will be used in a coupled transcription-translation system to generate truncated preproinsulins. These will be used to investigate the minimum size of nascent polypeptide chains that can be transfered across the ER membrane. Prohormone processing enzymes will be identified using a molecular approach: A cDNA library from cells exhibiting high levels of processing activity will be inserted into a retrovirus expression vector and recomtinant virus used to infect L cells in which preproSRIF cDNA has been stably integrated and which secrete only proSRIF, since they lack prohormone processing activity. Secretion of mature SRIF will be assayed using an antibody immunoblotting procedure; DNA will be isolated from positive cells and characterized. Identifying structural determinants that mediate processing of islet hormone presursors had direct relevance to understanding their synthesis and secretion and has important implications concerning the etiology of diabetes.