Bioactive peptides, key players in intercellular communication, are produced from inactive precursors by a series of highly conserved enzymes. A limited set of enzymes [prohormone convertases, carboxypeptidase E, glutaminyl cyclase, peptidylglycine alpha-amidating monooxygenase (RAM)] are specialized for the conversion of prohormones into active product peptides. These enzymes require the unique and progressively changing milieu of the secretory pathway. RAM, the only enzyme that produces alpha- amidated peptides, functions at the end of pathway, converting Gly-extended inactive precursors into bioactive amidated products. The RAM gene is essential - mice lacking RAM develop normally for about 12 days; edema then develops and few survive past embryonic day 14.5. Adult mice with a single functional RAM gene are viable and fertile, but exhibit altered glucose tolerance and altered behavioral responses to cocaine. RAM, a bifunctional enzyme, consists of PHM,a monooxygenase, and PAL,a unique lyase. Aim 1: Purified wild type and mutant PHM and PAL will be used to define both reaction mechanisms. An in situ amidation assay will be developed to assess the loading of metals onto PHM (Cu)and PAL (Fe, Zn,Ca). Aim 2: Adult PAM+/" mice will be used to determine the functions most sensitive to limited amidation activity. Using these assays,the effects of dietary or genetic deficiencies in Cu availability will be explored. The highly conserved cytosolic domain of RAM is essential for its entry into granules, endocytosis and the effects of RAM on cytoskeletal organization and gene expression. Aim 3: Covalent modifications of the cytosolic domain of RAMwill be identified and their effects on its functions explored. Aim 4: The role of phosphorylation and ubiquitination in the trafficking of membrane RAMwill be determined. Amidated peptides are key regulators of metabolism and homeostasis. These studies will identify the physiological systems most sensitive to deficits in production of these key signaling molecules.