The specific aims of the proposed research are: 1) to thoroughly investigate the protease(s) involved in the formation of AlphaMSH and Beta-endorphin farom ACTH and BetaLPH, respectively, in rat and bovine intermediate pituitaries 2) to characterize the enzyme(s) in the pituitary, and possibly other peptide secreting tissues, that is (are) responsible for the Alpha-amidation of AlphaMSH, and possibly other neuroendocrine peptides 3) to conduct a more in-depth investigation of the intermediate pituitary secretory granule-associated acetyltransferase including the purification, characterization of the molecular nature, tissue localization, and biosynthetic pathway of the enzyme The general approach involves the use of pituitary secretory granule extracts as a source for these enzyme activities, strategically radiolabeled peptides as substrates, and thorough identification of products primarily by reversed-phase high performance liquid chromatography (RP-HPLC). The purification of the acetyltransferase will be performed with a combination of classical techniques, dye-ligand and affinity chromatography, and HPLC. The long-term objectives involve determining how different tissues can regulate the post-translational processing of identical pro-ACTH/endorphin molecules to form tissue-specific collections of peptide hormones with different biological activities. Learning more about the enzymes responsible for the biosynthesis ACTH- and Beta-endorphin-related peptides will help us discover more about how secretagogues, both natural (dopamine and CRF) and synthetic (dexamethasone), can alter biosynthetic and/or secretion rates of these pituitary peptides.
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