SEROTONIN N-ACETYLTRANSFERASE IN THE PINEAL GLAND

松果体中的血清素 N-乙酰转移酶

• 批准号: 3235694
• 负责人: ARYAN Mangalam NAMBOODIRI
• 金额: $ 12.27万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235694
• 关键词: acyltransferase; antibody; circadian rhythms; complementary DNA; enzyme structure; gel electrophoresis; gene expression; genetic manipulation; high performance liquid chromatography; ion exchange chromatography; isoproterenol; laboratory mouse; laboratory rabbit; laboratory rat; melatonin; molecular cloning; monoclonal antibody; peptide chemical synthesis; photobiology; pineal body; protein biosynthesis; serotonin

The long term objective of this research program is the understanding of the molecular mechanisms involved in the postsynaptic regulation of neuronal function, using the catecholamine mediated regulation of serotonin N- acetyltransferase (NAT) activity in the rat pineal gland as a model. This enzyme regulates the concentration of the putative pineal hormone, melatonin, in the circulation. The significance of this study derives from 1) the wide spread role of postsynaptic regulatory processes in both normal and disordered development and maintenance of nervous system functions, as well as 2) the demonstrated role of melatonin as a regulatory hormone in many vertebrates and the likelihood that it has similarly important, but incompletely documented, functions in man. In order to study the molecular mechanisms involved in the regulation of NAT itself and, indirectly, melatonin synthesis, the following immediate aims are proposed: 1) produce antibodies against NAT, 2) purify NAT to homogeneity, 3) determine whether the synthesis of NAT protein is increased when NAT activity is stimulated at night and 4) clone NAT cDNA if NAT protein synthesis is found to be regulated by light-dark cycles. Pineal NAT in the rat exhibits a circadian rhythm with 50-100 fold increase in activity at night in the dark. The increase in NAT activity is mediated by an adrenergic-camp mechanism involving both transcriptional and translational events. The PI has recently succeeded in purifying NAT from rat pineal gland and plans to continue his studies to understand the molecular mechanisms involved in NAT regulation. A three tiered approach for the production of antibodies of different quality/specificity will be proposed in order to permit the orderly investigation of the molecular biology of NAT regulation. The hypothesis that the nocturnal increase in NAT activity involves increased synthesis of NAT protein will be tested by determining the amount of NAT protein during day and night; this will be done by immunotitration of NAT activity. In addition, the rate of incorporation of radiolabelled amino acids into NAT protein will be determined during day and night with the aid of NAT antibodies. If the synthesis of NAT protein is found to be increased at night, then efforts will be focused on cloning NAT cDNA in order to subsequently study in detail the mechanisms involved in the regulation of expression of NAT gene. If it is not found to be the case, then subsequent efforts will be directed towards the analysis to possible post-translational mechanisms of NAT regulation with the aid of antibodies.

该研究计划的长期目标是 了解参与的分子机制 神经元功能的突触后调节，利用 儿茶酚胺介导的血清素 N- 调节 大鼠松果体中的乙酰转移酶（NAT）活性 模型。 这种酶调节假定的浓度 松果体激素，褪黑激素，在循环中。 意义 这项研究源于 1) 突触后的广泛作用 正常和无序发展中的调节过程 和维持神经系统功能，以及2） 褪黑激素作为调节激素在许多方面的作用已被证明 脊椎动物及其具有同样重要的可能性，但是 不完全记录，在人体内发挥作用。 为了研究 NAT本身调控的分子机制 并间接合成褪黑激素，近期目标如下 建议：1) 生产抗 NAT 的抗体，2) 纯化 NAT 3）判断NAT蛋白是否合成 夜间刺激 NAT 活性时会增加，4) 克隆 NAT cDNA（如果发现 NAT 蛋白质合成受以下因素调节） 光暗循环。 大鼠松果体 NAT 呈现 50-100 的昼夜节律 夜间在黑暗中活动增加倍数。 NAT 的增加 活性由肾上腺素能营机制介导，涉及 转录和翻译事件。 PI 最近 成功从大鼠松果体中纯化 NAT 并计划 继续他的研究以了解分子机制 参与NAT监管。 三层方法 不同质量/特异性的抗体的生产将是 提议的目的是为了能够有序地调查 NAT 调节的分子生物学。 假设 夜间 NAT 活性的增加涉及 通过测定NAT的量来测试NAT蛋白 白天和晚上的蛋白质；这将通过免疫滴定来完成 NAT 活动。 此外，掺入率 放射性标记氨基酸转化为 NAT 蛋白后将被测定 白天和晚上在 NAT 抗体的帮助下。 如果 发现NAT蛋白的合成在夜间增加，则 工作重点将集中于克隆 NAT cDNA，以便 随后详细研究了其中涉及的机制 NAT基因表达的调控。 如果没有发现是 案例，那么后续的工作将针对分析 NAT 调节的可能翻译后机制 抗体的帮助。