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是 合成为较大的前体。 我们的长期目标是确定 前体在介导细胞内转运中的作用， 翻译后加工和激素的分泌。 PreproSRIF是 因为它是最简单的肽之一， 激素前体，仅含有一种生物活性肽和一种 蛋白水解加工位点，用于切除成熟激素。 使用 重组DNA技术，一系列突变的preproSRIF分子将被 构建来研究前体中的功能域。 定点诱变将用于改变细胞中的单个氨基酸。 蛋白水解加工位点;缺少确定区域的缺失突变体 的前肽将被构建，并且前肽将与 非分泌性蛋白质。 这些cDNA将被转染到异源细胞中， 细胞和分泌的参数进行了研究。 常见的排序和 酵母preproalpha因子和preproSRIF中的加工结构域将被 通过表达天然前原-SRIF cDNA和嵌合cDNA鉴定 在酵母中编码可变量的这些各自的前体。 cDNA 编码前胰高血糖素原的细胞将被引入不同的细胞类型， 研究了它们的翻译后加工和分泌。 在 与前SRIF原相反，前胰高血糖素原编码几种胰高血糖素相关的 肽，因此代表了下一个复杂的前体 处理. 翻译终止密码子将被引入前胰岛素原 cDNA将用于偶联转录-翻译系统， 产生截短的前胰岛素原。 这些将用于调查 可转移的新生多肽链的最小尺寸 ER膜。 激素原加工酶将使用 分子方法：来自表现出高水平的细胞的cDNA文库， 将处理活性插入逆转录病毒表达载体中 和用于感染L细胞的重组病毒，其中preproSRIF cDNA具有 已经稳定整合并且仅分泌proSRIF，因为它们缺乏 激素原加工活性。 将测定成熟SRIF的分泌 使用抗体免疫印迹程序; DNA将从 阳性细胞并进行表征。 确定结构决定因素， 胰岛激素前体的介导处理与 了解它们的合成和分泌， 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.