NOREPINEPHRINE BIOSYNTHESIS: ROLE OF ASCORBATE

抗坏血酸在儿茶酚胺生物合成中的作用 神经递质和神经肽激素，因此，总的来说 神经内分泌功能已被广泛认识。身份的识别 神经内分泌中复杂复杂的抗坏血酸再生系统 颗粒进一步阐明ASC在神经内分泌中的中枢作用 功能。尽管，最近的努力是针对 对ASC/ARS与儿茶酚胺相关功能的认识 神经递质与神经肽类激素的生物合成研究进展 努力一直受到复杂性和多样性的阻碍 细胞内和细胞外因素的影响。本文提出的工作是 重点了解ASC/ARS在儿茶酚胺中的作用 神经递质的生物发生与调节 两个主要的ASC依赖系统，多巴胺β-单加氧酶(DBM)和 细胞色素b561，在分子水平上。在我们计划的最初阶段 计划，我们将开发一种灵敏的、连续的分光光度分析方法 并将其扩展到测量神经内分泌的电子流量 以肾上腺嗜铬颗粒幽灵为模型的细胞色素b561颗粒 系统。在第二阶段，我们将开发各种基于ASC的化学品 探针，即替代电子供体、竞争性和自杀性抑制剂， 多底物/抑制剂、活性部位定向抑制剂和 光亲和标记法详细检测DBM的ASC部位。这个 从这些研究中获得的知识将被用来并扩展到研究 纯化重组细胞的化学特性及其特异性研究 系统，然后将其扩展到更复杂的Cyt b561介导的ARS 嗜铬颗粒幽灵。在第三阶段，我们将使用上述分析方法 以及基于ASC的化学探针，以检查额外的- 和细胞内因素对颗粒幽灵去甲肾上腺素生物合成的影响 和完整的嗜铬细胞颗粒，以了解分子 儿茶酚胺转运体， 膜ATPase，可溶的和膜结合的DBM和电子 转运蛋白，Cyt b561。这些研究的结果将产生 这些信息将对理解 ASC/ARS，在肾上腺素能神经分泌囊泡的动力学中。在……里面 此外，这些发现可能会扩展到肽能 神经分泌小泡，因为多肽处理终端酶， 肽酰胺化单加氧酶(PAM)，也是ASC依赖的。

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. Identification of a complex and intricate ascorbate regenerating system (ARS) in neuroendocrine granules further elaborate the central role of Asc in neuroendocrine functions. Although, the recent efforts have been directed towards the understanding of the functions of Asc/ARS in relation to catecholamine neurotransmitter and neuropeptide hormone biosynthesis, progress of these efforts has been hampered by the complexity and the diversity of the effects of intra- and extra-cellular factors. The work proposed herein is focused on the understanding of the role of Asc/ARS in catecholamine neurotransmitter biogenesis and regulation by systematic investigation of the two major Asc-dependent systems, dopamine beta-monooxygenase (DbM) and cytochrome b561, at the molecular level. In the initial phase of our program, we will develop a sensitive, continuous spectrophotometric assay for DbM and extend it to measure the electron flux into neuroendocrine granules via cyt b561 using adrenal chromaffin granule ghosts as a model system. In the second phase, we will develop various Asc-based chemical probes, i.e. alternate electron donors, competitive and suicide inhibitors, multi-substrates/inhibitors, active site directed inhibitors, and photoaffinity labels to examine the Asc site of DbM in detail. The knowledge gained by these studies will be used and extended to examine the chemistry and the specificity of cyt b561 using purified reconstituted systems and then extend it to more complex cyt b561-mediated ARS in chromaffin granule ghosts. In the third phase, we will use the above assay as well as the Asc-based chemical probes to examine the effect of extra- and intra-cellular factors on norepinephrine biosynthesis in granule ghosts and intact chromaffin granules, in order to understand the molecular mechanisms and interrelations between the catecholamine transporter, membrane ATPase, both soluble and membrane bound DbM and the electron transport protein, cyt b561. The results of these studies will yield information which will be valuable in understanding the crucial role of Asc/ARS, in the dynamics of adrenergic neurosecretory vesicles. In addition, these findings could probably be extended into the peptidergic neurosecretory vesicles, since the peptide processing terminal enzyme, peptidyl amidating monooxygenase (PAM), is also Asc-dependent.