IN VITRO BIOSYNTHESIS OF PANCREATIC POLYPEPTIDE HORMONES

从朗格汉斯胰岛分离的信使RNA将被翻译成 无细胞蛋白质合成系统研究生物合成和 多个胰高血糖素前体的翻译后修饰 生长抑素。三个前高血糖素分子将被表征为 双向凝胶电泳法、多肽图谱和自动化 微测序技术，以确定结构关系 每一个成熟的胰高血糖素的前体。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， 将对磷酸化和/或脂质添加进行研究。试管苗 比较胰岛和脑组织中生长抑素的生物合成 以确定(A)多个前生长抑素分子是否也 在大脑中合成(B)与胰岛前体相似。在……里面 平行研究，两个不同种类的胰岛的特定cdna克隆 前生长抑素mRNA将被用来探测脑mRNA，以分析 不同生长抑素基因的表达。完整的氨基酸 脑前生长抑素(S)的序列将用 重组DNA方法学。这些实验将证明是否有 生长抑素基因的组织特异性表达。三种多肽 与鱼、鼠和人的保守区相对应 前生长抑素已被合成。这些多肽中的每一个都会 用于生产针对特定区域的特异性抗体 前列生长抑素。抗血清将被用来研究 大鼠胰岛肿瘤细胞株分泌生长抑素过程中的前肽。 免疫沉淀、免疫荧光和电子技术 使用胶体金的显微镜免疫细胞化学定位 用来确定前生长抑素或前体片段是否 在细胞内降解或与成熟的激素一起分泌。 用针对前片段的抗血清获得的结果将是 与使用抗生长抑素抗体的患者相关。是否应该有证据 除了成熟的荷尔蒙外，还可以分泌前肽， 这可能表明这种多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.