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.

超过一半的已知神经内分泌肽是 α-酰胺化的 在早期的所有情况下，这种结构特征对于受体来说是必需的 识别和信号转导。 α-酰胺化反应由以下物质催化 肽基甘氨酸 α-酰胺化单加氧酶 (PAM)，双功能酶 定位于分泌颗粒。 羟化酶和裂解酶活性 PAM 依次催化 α-酰胺化的最后两个步骤。 这 羟基化步骤，由肽基甘氨酸α-羟基化催化 单加氧酶 (PHM) 具有限速作用，可以确定总体 α-酰胺化肽的生产。 最近确定， PHM 的活性通过共价修饰来调节，从而增加 酶的 V max 。 此修改是为了响应 长期持续使用双硫仑治疗 似乎是由通常控制 PHM 的生理过程介导的 体内。 该研究有三个具体目标。 第一个目标是定义 增加 PHM V max 的修饰的化学性质 对双硫仑治疗的反应。 来自对照和双硫仑的酶 处理过的动物将被蛋白水解消化并通过 HPLC 分级。 肽产品及其氨基酸成分将使用 基质辅助激光解吸、飞行时间质谱仪和 配备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。 第二个目的是研究调节机制 使用培养的神经和内分泌细胞进行修饰。 细胞培养 实验将确定双硫仑代谢物在体内的作用 PHM 的反应并研究生理学的可能性 PHM的修饰在神经诱导的神经元分化中发挥作用 pC12 细胞中的生长因子。 第三个目标是确定角色 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 将在肾上腺切除的大鼠和培养物中检查 PHM 的 V max 新生大鼠心房肌细胞。 本研究的总体目标是确定机制 控制神经肽中限速酶的活性 生物合成。 这项研究还将带来对行动的新见解 双硫仑。 尽管双硫仑已被广泛用作酒精 令人惊讶的是，目前对其分子了解甚少 机制。 正如双硫仑所表明的，这个问题的重要性日益增加 有望治疗获得性免疫缺陷综合征。