NOREPINEPHRINE BIOSYNTHESIS--ROLE OF ASCORBATE

去甲肾上腺素生物合成——抗坏血酸的作用

• 批准号: 6180408
• 负责人: KANDATEGE WIMALASENA
• 金额: $ 15.97万
• 依托单位: WICHITA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180408
• 关键词: active sites; ascorbate; chromaffin cells; copper; cytochrome b; dopamine beta monooxygenase; electron probe spectrometry; electron transport; enzyme activity; enzyme mechanism; enzyme substrate; membrane transport proteins; metalloproteins; molecular dynamics; neuroendocrine system; neurotransmitter biosynthesis; norepinephrine; oxidoreductase inhibitor; protein purification; ultraviolet spectrometry

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.

中枢和中枢神经递质儿茶酚胺和神经肽 周围神经激素系统可能与临床疾病有关 例如高血压、心脏异常和神经功能障碍 比如精神分裂症和其他精神疾病。因此， 了解体内儿茶酚胺神经递质和 分子水平的神经肽激素生物合成将很重要 为了了解这些疾病的病因以及最终的结果 开发有效的治疗剂。抗坏血酸的作用 (Asc) 参与儿茶酚胺神经递质和 神经肽激素，因此，在整体神经内分泌功能中 得到了很好的认可。由于有大量的神经内分泌分泌小泡 肾上腺髓质或其他内分泌腺不会转移 Asc 囊泡膜、跨膜血红素蛋白、细胞色素 b561 建议负责转移必要的减少 儿茶酚胺生物合成酶、多巴胺β-的等效物 单加氧酶（DbM）和神经肽加工末端酶， 肽基 α-羟基化单加氧酶 (PHM)，来自胞质池 上升。尽管最近的努力已针对 了解分子中这种复杂的电子转移过程 与儿茶酚胺和神经肽生物合成相关的水平， 这些努力的进展因复杂性和 细胞内和细胞外因素影响的多样性。整体 我们计划的目标是定义分子机制 还原当量从胞质 Asc 转移到内部 通过仔细检查氧化还原来确定神经内分泌颗粒 途径中各种蛋白质的相互作用。我们提议 通过检查还原剂的相互作用来实现这一目标， 阴离子酶激活剂和其他具有 DbM 的底物，尤其是在 与使用各种合成的活性位点铜氧化还原中心的关系 探针和光谱技术，并将类似的研究扩展到 PHM。 与此同时，我们将纯化并进一步表征新发现的 酸性铜蛋白作为细胞色素 b561 和 DbM 之间的氧化还原介体 （和PHM）。我们将研究单向的分子机制 使用 b561 穿过颗粒膜的电子传输 生物化学和生物物理技术。最后所有的信息都会 结合起来提出一个体内减少两者的分子模型 DbM 和 PHM。