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.

所有已知的神经内分泌肽中有一半以上是α-酰胺化的， 在早期所有情况下，这种结构特征对于受体 识别和信号转导。 α-酰胺化由以下物质催化： 肽酰甘氨酸α-酰胺化单加氧酶，双功能酶 定位于分泌颗粒中。 羟化酶和裂解酶活性 PAM依次催化α-酰胺化中的最后两个步骤。 的 羟基化步骤，由肽基甘氨酸α-羟基化催化 单加氧酶（PHM）是限速的，可以决定整体的 生产α-酰胺化肽。 最近确定， PHM的活性通过共价修饰来调节， 酶的Vmax。 这种修改是为了响应 用双硫仑治疗，持续很长一段时间， 似乎是由通常控制PHM的生理过程介导的 in vivo. 这项研究有三个具体目标。 第一个目标是定义 提高PHM的V max的改性的化学性质 对双硫仑治疗的反应。 来自对照和双硫仑的酶 处理的动物将被蛋白水解消化并通过HPLC分级。 肽产品及其组分氨基酸将使用 基质辅助激光解吸，飞行时间质谱仪和 配备有快速原子轰击的双聚焦质谱仪， 结构的电喷雾电离和碰撞诱导解离 分析. 第二个目的是研究介导 使用培养的神经和内分泌细胞进行修饰。 细胞培养 实验将确定双硫仑代谢物在他体内的作用。 PHM的反应，并研究生理性 PHM的修饰在神经诱导的神经元分化中起作用 pC 12细胞中的生长因子。 第三个目标是确定 PHM对双硫仑的持续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 将在肾上腺切除大鼠和培养物中检查PHM的Vmax 新生大鼠心房肌细胞。 本研究的总体目标是确定 控制神经肽中限速酶的活性 生物合成 这项研究也将导致对行动的新见解 双硫仑 尽管双硫仑已被公认为一种酒精， 令人惊讶的是，目前对其分子结构知之甚少。 机制等 随着双硫仑的出现， 获得性免疫缺陷综合征的治疗前景。