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PEPTIDE ALPHA AMIDATION--MECHANISMS FOR REGULATION

肽α酰胺化——调节机制

基本信息

批准号：
2431276
负责人：
GREGORY P MUELLER
金额：
$ 24.33万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-06-01 至 2000-05-31
项目状态：
已结题

项目摘要

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 is 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 is 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 is 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.

在所有已知的神经内分泌肽中，超过一半是α-酰胺化的和在早期的所有病例中，这种结构特征对受体是必不可少的。识别和信号转导。α-酰胺化的催化作用是双功能酶甘氨酸氨基单加氧酶(PAM) 定位于分泌颗粒中。羟基酶和裂解酶活力的测定 PAM依次催化α-酰胺化反应的最后两步。这个羟化步骤，由氨基甘氨酸α-羟基化催化单加氧酶(PHM)，是限速的，可以决定总体 α-酰胺化多肽的生产。最近确定， PHM的活性是通过共价修饰调节的，这种修饰增加了酶的Vmax。这一修改是为了响应用双硫仑治疗，持续很长一段时间似乎是通过正常控制PHM的生理过程来调节的在活体内。这项研究有三个具体目标。第一个目标是定义提高PHM的Vmax的修饰的化学性质对双硫兰治疗的反应。来自对照和二硫胺的酶经过处理的动物将被蛋白质降解，并通过高效液相色谱进行分级。多肽产品及其组成的氨基酸将使用基质辅助激光解吸、飞行时间质谱仪和配备快原子轰击的双聚焦质谱计和结构的电喷雾电离和碰撞诱导解离分析。第二个目标是研究调节使用培养的神经和内分泌细胞进行修饰。细胞培养实验将确定双硫兰代谢物在他体内的作用 PHM的反应，并调查一种生理学 PHM的修饰在神经诱导神经元分化中的作用 PC12细胞中的生长因子。第三个目标是确定肾上腺皮质激素在双硫仑持续体内反应中的作用治疗。糖皮质激素在调节血管紧张素转换酶长期变化中的作用将在去肾上腺的大鼠和培养的大鼠身上检测PHM的Vmax 新生大鼠心房肌细胞。这项研究的总体目标是定义控制神经肽中一种限速酶的活性生物合成。这项研究还将带来对这一行动的新见解双硫仑。尽管双硫仑已被广泛用作酒精威慑力，令人惊讶的是，目前人们对其分子知之甚少机制。正如双硫胺所显示的，这个问题的重要性增加了在治疗获得性免疫缺陷综合征方面有希望。