IN VITRO BIOSYNTHESIS OF INSULIN AND GLUCAGON

点击翻译按钮获取中文摘要

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.