CHOLINE ACETYLTRANSFERASE

本项目的总体目标是阐明 胆碱乙酰转移酶在调节 神经递质乙酰胆碱的合成。 之一 该项目的主要具体目标是获得cDNA 对应于ChAT信使RNA。 新的cDNA文库已经被 在λ gt10和λ Zap中使用来自 胆碱能细胞系CHP 134。 这些图书馆将被筛选 与本实验室制备的几种抗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肽对应 约占人类ChAT一级序列的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用于表达 酶用于机械研究。 已经表明，ChAT可以被磷酸化， 大鼠脑Ca/钙调蛋白激酶和蛋白激酶C。 研究 也将集中在确定磷酸化的影响， 酶的性质。 这些研究将包括分析 钙/钙调蛋白激酶磷酸化酶的动力学 激活蛋白和蛋白激酶存在和不存在 C磷酸化酶。 鼠脑酶的两种pI形式 分离以确定它们是否代表天然和磷酸化 酵素 培养细胞的研究将用于呈现 体内ChAT磷酸化的证据，并评估其作用 磷酸化对乙酰胆碱合成的影响。 分离和 含有活性位点半胱氨酸、精氨酸 和组氨酸残基。 鉴定这些 酶中的残基将为 确定活动地点，并将作为地点的联络点- 特异性诱变

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.