SEROTONIN N-ACETYLTRANSFERASE IN THE PINEAL GLAND

这项研究计划的长期目标是 对相关的分子机制的理解 神经功能的突触后调节，使用 儿茶酚胺对5-羟色胺的调节作用 大鼠松果体中乙酰转移酶(NAT)的活性 模特。这种酶调节推定的 松果体激素，褪黑激素，在循环中。的重要意义。 这项研究源于1)突触后的广泛作用 正常和无序发育的调控过程 以及神经系统功能的维护，以及2) 褪黑素作为一种调节激素在许多疾病中的作用 脊椎动物和它同样重要的可能性，但 不完全有文献记载，在人类中的功能。为了更好地研究 NAT自身调控的分子机制 间接地，褪黑素的合成，有以下直接的目的 提出：1)制备抗NAT抗体；2)纯化NAT以 同质性，3)决定NAT蛋白的合成是否 在夜间刺激NAT活动和克隆时会增加 NAT cDNA如果发现NAT蛋白质合成受 光明-黑暗的循环。 大鼠松果体NAT呈50-100的昼夜节律 夜间在黑暗中的活动增加了一倍。NAT的增加 活性是由肾上腺素能cAMP机制介导的， 无论是转录事件还是翻译事件。公安部最近 成功从大鼠松果体中提纯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，用于 将生产不同质量/特异性的抗体 提议的目的是为了允许对 NAT调控的分子生物学。这一假设认为 夜间NAT活性的增加涉及到增加的合成 将通过确定NAT的量来测试NAT蛋白 蛋白质在白天和晚上；这将通过免疫滴定完成 NAT活动。此外，成立公司的比率 放射性标记氨基酸转化为NAT蛋白将被测定 在NAT抗体的帮助下，白天和晚上。如果 NAT蛋白合成在夜间增加，然后 我们将致力于NAT基因的克隆，以便 随后详细研究了该系统所涉及的机制。 NAT基因的表达调控。如果没有发现它是 案例，那么后续的工作将指向分析 关于NAT调节的可能的翻译后机制 在抗体的帮助下。

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.