SEROTONIN N-ACETYLTRANSFERASE IN THE PINEAL GLAND

这项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，使用 儿茶酚胺介导的5-羟色胺N- 乙酰转移酶（NAT）活性在大鼠松果体作为一个 模型 这种酶调节假定的 松果体激素，褪黑激素，在血液循环中。 的意义 这项研究源于1）突触后神经元的广泛作用， 正常和无序发育的调节过程 和维持神经系统功能，以及2） 褪黑激素作为调节激素的作用在许多 脊椎动物和可能性，它有同样重要的，但 不完全记录，功能的人。为了研究 参与NAT自身调节的分子机制 以及，间接地，褪黑激素的合成， 提出：1）产生针对NAT的抗体，2）纯化NAT， 同质性，3）确定是否合成NAT蛋白 当NAT活性在夜间受到刺激时， 如果发现NAT蛋白质合成受 明暗循环 大鼠的松果体NAT表现出50-100的昼夜节律 在黑暗中的夜间活动增加一倍。 NAT的增加 活性由肾上腺素能cAMP机制介导， 包括转录和翻译事件。 PI最近 成功地从大鼠松果体中纯化了NAT，并计划 继续他的研究以了解 参与NAT监管。 一个三层的方法， 不同质量/特异性的抗体的产生将是 建议，以允许有序的调查， NAT调节的分子生物学。 的假设 夜间NAT活性的增加涉及增加的 通过测定NAT的量来检测NAT蛋白 蛋白质在白天和晚上;这将通过免疫滴定法进行 NAT活动。 此外， 将测定NAT蛋白中的放射性标记氨基酸 在NAT抗体的帮助下， 如果 发现NAT蛋白的合成在夜间增加， 将集中精力克隆NAT cDNA， 随后，详细研究了 NAT基因的表达调控。 如果没有发现 案例，随后的工作将针对分析 NAT调节的可能的翻译后机制， 抗体的帮助。

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.