CHOLINE ACETYLTRANSFERASE

本项目的总体目标是阐明 胆碱乙酰转移酶在细胞周期中的调控 神经递质乙酰胆碱的合成。其中一个 这个项目的主要具体目标是获得一个 对应于聊天信使RNA。新的cdna文库已经被 在lambda gt10和lambda zap中使用来自 胆碱能细胞系CHP134。将对这些图书馆进行筛选 本实验室还制备了几种抗Chat抗血清 与基于蛋白质制备的寡核苷酸探针一样 在这个实验室里产生的序列。到目前为止，我们已经测序了 总共有10个溴化氰和胰蛋白酶多肽对应 到人类聊天的主要序列的大约20%。 分离的cDNA克隆将用于研究ChAT的调控 在培养细胞中的诱导以及对 用于机械学研究的酶。 已经证明，聊天可以通过一种 大鼠脑组织中的钙/钙调蛋白激酶和蛋白激酶C。研究 还将重点确定磷酸化对 该酶的性质。这些研究将涉及一项分析 钙/钙调蛋白激酶磷酸化酶动力学的研究 激活蛋白和蛋白激酶的存在与缺失 C磷酸化酶。大鼠脑酶的两种等电点形式 被分离以确定它们是否代表天然和磷 酵素。培养细胞的研究将被用来呈现 ChAT在体内磷酸化的证据及其效果评估 磷酸化对乙酰胆碱合成的影响。与世隔绝和 含有半胱氨酸、精氨酸活性部位的多肽的序列分析 组氨酸残留物也在计划中。这些人的身份 酶中的残留物将为 确定活动地点，并将作为地点的焦点- 特定的突变作用。

The overall objective of this project is to elucidate the role of the enzyme choline acetyltransferase in the regulation of the synthesis of the neurotransmitter acetylcholine. One of the primary specific goals of this project is to obtain a cDNA corresponding to ChAT messenger RNA. New cDNA libraries have been prepared in lambda gt10 and lambda Zap using polyA mRNA from the cholinergic cell line CHP134. These libraries will be screened with several anti-ChAT antisera prepared in this laboratory as well as with oligonucleotide probes prepared on the basis of protein sequence generated in this laboratory. To date we have sequenced a total of 10 cyanogen bromide and tryptic peptides corresponding to approximately 20% of the primary sequence of human ChAT. Isolated cDNA clones will be used to study the regulation of ChAT induction in cultured cells as well as for the expression of the enzyme for mechanistic studies. It has been shown that ChAT can be phosphorylated by a Ca/calmodulin kinase and protein kinase C from rat brain. Studies will also focus on determining the effect of phosphorylation on the properties of the enzyme. These studies will involve an analysis of the kinetics of Ca/calmodulin kinase phosphorylated enzyme in the presence and absence of activator protein and protein kinase C phosphorylated enzyme. Two pI forms of the rat brain enzyme will be isolated to determine whether they represent native and phospho enzyme. Studies with cultured cells will be used to present evidence for ChAT phosphorylation in vivo and to assess the effect of phosphorylation on acetylcholine synthesis. The isolation and sequencing of peptides containing active site cysteine, arginine and histidine residues are planned. The identification of these residues in the enzyme will provide the foundation with which to identify the active site and will serve as a focal point for site- specific mutagenesis.