IN VITRO BIOSYNTHESIS OF PANCREATIC POLYPEPTIDE HORMONES

从胰岛中分离的信使RNA将被翻译成 无细胞蛋白质合成系统，用于研究生物合成， 胰高血糖素的多种前体的翻译后修饰， 生长抑素 三种胰高血糖素原分子的特征在于： 双向凝胶电泳、肽图谱和自动化 微测序技术，以确定结构关系， 每种都是成熟胰高血糖素的前体。 前胰高血糖素原经历不同的 体外翻译后修饰和可能的糖基化， 将研究磷酸化和/或脂质添加。 体外 将比较来自胰岛和脑mRNA的生长抑素的生物合成 为了确定（a）多种前生长抑素原分子是否也 在脑中合成（B）与胰岛前体的相似性。 在 平行研究，两种不同胰岛的特异性cDNA克隆 前生长抑素原mRNA将用于探测脑mRNA，以分析 不同生长抑素基因的表达。 完整氨基酸 脑前生长抑素原的顺序将使用 重组DNA方法。 这些实验将证明 生长抑素基因的组织特异性表达。 三种多肽 对应于鱼类、大鼠和人类的保守前区 已经合成了前生长抑素原。 这些多肽中的每一种将 用于生产针对特定区域的特异性抗体， 生长抑素原 抗血清将用于研究 前肽在大鼠胰岛肿瘤细胞系生长抑素分泌过程中。 免疫沉淀、免疫荧光和电镜技术 显微镜免疫细胞化学定位使用胶体金将是 用于确定是否促生长素抑制素或前体的片段， 在细胞内降解或与成熟激素一起沿着分泌。 用抗前片段的抗血清获得的结果将在 与使用抗生长抑素抗体的患者相关。 应该有证据 除了成熟激素外，还可分泌前肽， 这可能表明这些肽可能具有生理功能。

Messenger RNA isolated from islets of Langerhans will be translated in a cell-free protein synthesizing system to study the biosynthesis and post-translational modifications of multiple precursors to glucagon and somatostatin. Three proglucagon molecules will be characterized by two-dimensional gel electrophoresis, peptide mapping and automated microsequensing techniques, to determine the structural relationship of each precursor to mature glucagon. The preproglucagons undergo different post-translational modifications in vitro and possible glycosylation, phosphorylation and/or lipid addition will be investigated. The in vitro biosynthesis of somatostatin from islet and brain mRNA's will be compared to determine (a) if multiple preprosomatostatin molecules are also synthesized in the brain (b) the similarity to the islet precursors. In parallel studies, specific cDNA clones to two different species of islet preprosomatostatin mRNA will be used to probe brain mRNA to analyze the expression of distinct somatostatin genes. The complete amino acid sequence of brain preprosomatostatin(s) will be determined using recombinant DNA methodology. These experiments will show if there is tissue-specific expression of the somatostatin genes. Three polypeptides corresponding to conserved proregions of fish, rat and human preprosomatostatin have been synthesized. Each of these polypeptides will be used in the production of specific antibodies to defined regions of prosomatostatin. The antisera will be used to study the fate of the propeptide during somatostatin secretion from a rat islet tumor cell line. Techniques of immunoprecipitation, immunofluorescence and electron microscopy immunocytochemical localization using colloidal gold will be employed to determine if prosomatostatin or fragments of the precursor are degraded intracellularly or are secreted along with the mature hormone. Results obtained with antisera directed against profragments will be correlated with those using antisomatostatin antibodies. Should evidence be found for secretion of propeptides, in addition to the mature hormone, this might suggest such peptides could have physiological functions.