More than half of all known neuroendocrine peptides are alpha-amidated and in early all cases, this structural feature is essential for receptor recognition and signal transduction. alpha-Amidation is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM), bifunctional enzyme localized in secretory granules. The hydroxylase and lyase activities of PAM sequentially catalyze the final two steps in alpha-amidation. The hydroxylation step, catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM), is rate limiting and can determine the overall production of alpha-amidated peptides. It was recently determined that the activity of PHM is regulated through a covalent modification that increases the V max of the enzyme. This modification, which occurs in response to treatment with disulfiram, is sustained over a long period of time and appears to be mediated by a physiologic process that normally controls PHM in vivo. The research has three specific aims.
The first aim i s to define the chemical nature of the modification that increases the V max of PHM in response to disulfiram treatment. Enzyme from control and disulfiram treated animals will be digested proteolytically and fractionated by HPLC. Peptide products and their component amino acids will be analyzed using a matrix-assisted laser desorption, time-of-flight mass spectrometer and a double focusing mass spectrometer equipped with fast atom bombardment and electrospray ionization and collision-induced dissociation for structural analysis.
The second aim i s to investigate the mechanism that mediates the modification using cultured neural and endocrine cells. Cell culture experiments will determine the role of disulfiram metabolites int he response of PHM and investigate the possibility that a physiologic modification of PHM has a role in neuronal differentiation induced by nerve growth factor in pC12 cells.
The third aim i s to determine the role of adrenal status in the sustained in vivo response of PHM to disulfiram treatment. The role of glucocorticoids in regulating long term changes in the V max of PHM will be examined in adrenalectomized rats and in cultures of neonatal rat atrial myocytes. The overall objective for this research is to define the mechanism that controls the activity of a rate limiting enzyme in neuropeptide biosynthesis. This research will also lead to new insights into the action of disulfiram. Despite the established use of disulfiram as an alcohol deterrent, surprisingly little is currently known about its molecular mechanisms. The importance of this issue increases as disulfiram shows promise in t he treatment of acquired immune deficiency syndrome.
