IN VITRO BIOSYNTHESIS OF INSULIN AND GLUCAGON

钓鱼岛和鳕鱼胰岛中含Poly A总RNA的提取 朗格汉斯将在小麦胚芽中翻译无细胞蛋白质合成 用于表征假定的胰岛素原和胰高血糖素原并利用其的系统 该系统用于检验“信号假说”的预测。这些前兆 分子将通过各种不同的翻译产物从总的翻译产物中识别出来 分析程序，包括特异性抗体沉淀 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和多肽图谱。完整的氨基 这些分子的N-末端信号序列的酸性序列将是 使用自动测序技术确定。哺乳动物“被剥离” 微粒体膜将以共翻译的方式添加到无细胞系统中 对原胰高血糖素的炮制和分离产品进行鉴定。 信号肽的命运和最终的亚细胞定位(S) 从胰岛素原和胰升糖素原中去除后， 通过分离外源添加的微体囊泡进行研究 翻译系统。将对加工和分离的产品进行表征 经十二烷基硫酸钠-凝胶电泳法、凝胶过滤和自动Edman降解。如果 信号肽不会立即降解，其随后的定位将 用细胞分级法测定，并将进行实验以 确定是否可能分泌一个可能完整的信号肽。

Total poly A containing mRNA isolated from Angler Fish and Cod islets of Langerhans will be translated in the wheat germ cell-free protein synthesizing system to characterize putative preproinsulin and preproglucagon and to utilize this system for testing predictions of the "signal hypothesis". These precursor molecules will be identified from the total translation products by a variety of analytical procedures including specific antibody precipitation SDS-polyacrylamide gel electrophoresis and peptide mapping. The complete amino acid sequence of the N-terminal signal sequences of these molecules will be determined using automatic sequencing techniques. Mammalian "stripped" microsomal membranes will be added to the cell-free system cotranslationally to determine and identify the processed and segregated products of proglucagon. The fate and ultimate sub-cellular localization of the signal peptide(s) following its removal from preproinsulin and preproglucagon, will be investigated by isolating the exogenously added microsomal vesicles from the translation system. The processed and segregated products will be characterized by SDS-gel electrophoresis, gel filtration and automatic Edman degradation. If the signal peptide is not immediately degraded, its subsequent localization will be determined using cell-fractionation and experiments will be performed to determine if a possibly intact signal peptide might be secreted.