This application describes a program of research in peptide hormone endocrinology that spans both considerations of hormone biogenesis, action and degradation and the disciplines of biochemistry, chemistry, cell biology and physiology. Emphasis in this application is placed on the bioactive peptides of the endocrine pancreas (insulin, glucagon, somatostatin and pancreatic polypeptide) and on the precedents that may be gleaned by the study of these hormones in concern. While several different projects are described, they are joined by a common focus in chemical structure, biochemical specificity and molecular interactions.
Specific aims i nclude (a) analysis of the specificity of prohormone conversion at paired and single dibasic amino acid conversion sites by model enzymes and tissue proteases, (b) determination of the structures of abnormal products of peptide hormone genes and of the causes of hyper(pro)insulinemia in selected cases where insulin gene structure appears to be normal, (c) analysis of detailed structure-function relationships and conformational changes involved in the combination of insulin with its receptor, (d) examination of the kintic and chemical heterogeneity of insulin receptors expressed in hepatic tissue, (e) determination of the molecular basis of multiple glucagon receptor populations on hepatocytes and hepatic membranes, (f) analysis of the causes and consequences of glucagon antagonism and desensitization in isolated hepatocytes, (g) determination of the biochemical basis for multiple ligand-receptor states that occur subsequent to peptide hormone-receptor interactions, (h) evaluation of the intracellular sites and biochemical correlates of peptide hormone metabolism by target tissues, (i) determintion of the existence of peptide hormone metabolites in the circulation in man, and (j) analysis of potential regulatory mechanisms that modulate ligand-receptor interactions and peptide hormone degradation in hepatic tissue. Methods involve isolated cell and membrane incubation, tissue extraction for identification of peptides and modifying enzymes, kinetic measurements of hormone-receptor interactions, biochemical and chemical analysis of detergent-solubilized receptors, radiolabeling of peptide hormones and their analogs, peptide and protein purification and analysis by molecular sieve and reverse-phase chromatography and by other methods of protein chemistry, and chemical synthesis of peptides and peptide analogs for use as receptor probes. While these studies relate most directly to investigation of diabetes and other diseases of the endocrine pancreas, the long-term objective relates more broadly to understanding how endocrine regulations leads to metabolic homeostasis and how variations in control can lead to inappropriate metabolic regulation in man.
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