NOREPINEPHRINE BIOSYNTHESIS--ROLE OF ASCORBATE

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The catecholamine neurotransmitters and neuropeptides in the central and peripheral neurohormonal system may be implicated in clinical disorders such as hypertension, cardiac abnormalities and neurological dysfunctions like schizophrenia and other mental illnesses. Therefore, the understanding of the in vivo catecholamine neurotransmitter and neuropeptide hormone biosynthesis at the molecular level will be important for the understanding of the etiology of these diseases and eventual development of effective therapeutic agents. The role of ascorbic acid (Asc) in the biosynthesis of both catecholamine neurotransmitters and neuropeptide hormones and thus, in overall neuroendocrine functions has been well recognized. Since numerous neuroendocrine secretory vesicles of the adrenal medullae or other endocrine glands do not transfer Asc across the vesicle membrane, the transmembrane hemoprotein, cytochrome b561, is proposed to be responsible for transferring the necessary reducing equivalents for both the catecholamine biosynthetic enzyme, dopamine beta- monooxygenase (DbM), and the neuropeptide processing terminal enzyme, peptidyl alpha-hydroxylating monooxygenase (PHM), from the cytosolic pool of Asc. Although, recent efforts have been directed towards the understanding of this intricate electron transfer process at the molecular level, in relation to catecholamine and neuropeptide biosynthesis, progress of these efforts has been hampered by the complexity and the diversity of the effects of intra-and extra-cellular factors. The overall objective of our program is to define the molecular mechanism of the transfer of reducing equivalents from the cytosolic Asc to the interior of the neuroendocrine granule by careful examination of the redox interactions of various proteins in the pathway. We are proposing to achieve this objective by examining the interaction of the reductant, anion enzyme activators, and other substrates with DbM especially in relation to the active site copper redox centers using various synthetic probes and spectroscopic techniques and to extend similar studies to PHM. In parallel, we will purify and further characterize the newly identified acidic cooper protein as a redox mediator between cytochrome b561 and DbM (and PHM). We will examine the molecular mechanism of the unidirectional electron transport across the granule membrane through b561 using biochemical and biophysical techniques. Finally all the information will be combined to propose a molecular model for the in vivo reduction of both DbM and PHM.