IN VITRO BIOSYNTHESIS OF PANCREATIC POLYPEPTIDE HORMONES

点击翻译按钮获取中文摘要

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.