PEPTIDE ALPHA AMIDATION--MECHANISMS FOR REGULATION

肽α酰胺化——调节机制

• 批准号: 2273316
• 负责人: GREGORY P MUELLER
• 金额: $ 24.4万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2273316
• 关键词: PC12 cells; SDS polyacrylamide gel electrophoresis; amidation /deamidation; chemical kinetics; disulfiram; electrospray ionization mass spectrometry; enzyme activity; enzyme mechanism; glucocorticoids; high performance liquid chromatography; hormone regulation /control mechanism; hydroxylation; lyase; mass spectrometry; neurotrophic factors; peptides; proteolysis; tissue /cell culture; western blottings

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.

所有已知神经内分泌肽的一半以上是α腺体的，并且 在所有情况下，这种结构特征对于受体至关重要 识别和信号转导。 α-刺激被催化 肽基甘氨酸α的单加氧酶（PAM），双功能酶 本地分泌颗粒。 羟化酶和裂解酶活性 PAM依次催化α-增强的最后两个步骤。 这 羟基化步骤，由肽基甘氨酸α-羟基催化 单加氧酶（PHM）是限制速率，可以确定总体 产生α增强的肽。 最近确定 PHM的活性通过共价修改来调节，该修饰增加 酶的V Max。 这种修改是为了响应于 用二硫仑治疗，长期以来维持 似乎是由通常控制PHM的生理过程介导的 体内。 该研究具有三个具体目标。 第一个目的是定义 修饰的化学性质，增加了PHM的V max in 对二硫仑治疗的反应。 来自对照和二硫仑的酶 治疗的动物将被蛋白水解消化，并通过HPLC分离。 将使用A分析肽产品及其成分氨基酸 基质辅助激光解吸，飞行时间质谱仪和A 双聚焦质谱仪配备了快速原子轰击和 电喷雾电离和碰撞诱导的解离以进行结构 分析。 第二个目的是研究介导的机制 使用培养的神经和内分泌细胞进行修饰。 细胞培养 实验将确定二硫仑代谢产物的作用 PHM的反应并研究生理的可能性 PHM的修饰在神经诱导的神经元分化中起作用 PC12细胞中的生长因子。 第三个目的是确定 PHM持续的体内反应中的肾上腺状态 治疗。 糖皮质激素在调节长期变化中的作用 PHM的V Max将在肾上腺切除大鼠和培养物中进行检查 新生儿大鼠心房肌细胞。 这项研究的总体目的是定义一种机制 控制速率限制酶在神经肽中的活性 生物合成。 这项研究还将导致对行动的新见解 二硫仑。 尽管已建立了二硫仑作为酒精的使用 威慑，令人惊讶的是目前对其分子知之甚少 机制。 随着杜富拉姆的表明，这个问题的重要性增加了 有望治疗获得的免疫缺陷综合征。