CHOLINE ACETYLTRANSFERASE

胆碱乙酰转移酶

基本信息

批准号：
3116583
负责人：
Louis B. Hersh
金额：
$ 12.62万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-07-01 至 1992-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3116583
关键词：
acetylcholine calcium metabolism calmodulin choline acetyltransferase complementary DNA enzyme mechanism enzyme structure genetic library laboratory rat messenger RNA molecular cloning neurotransmitter metabolism nucleic acid probes phosphorylation protein kinase C protein sequence tissue /cell culture

项目摘要

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.

该项目的总体目的是阐明酶胆碱乙酰转移酶调节神经递质乙酰胆碱的合成。中的一个该项目的主要特定目标是获得cDNA 对应于聊天信使RNA。新的cDNA图书馆已经在Lambda GT10和Lambda Zap中使用Polya mRNA从胆碱能细胞系CHP134。这些库将被筛选在该实验室中还准备了几个反聊天的抗血清与基于蛋白质制备的寡核苷酸探针一样该实验室产生的序列。迄今为止我们已经测序了总共10种氰基溴和胰蛋白酶肽相应大约有20％的人聊天序列。孤立的cDNA克隆将用于研究聊天的调节培养细胞的诱导以及表达机械研究的酶。已经表明，聊天可以通过来自大鼠脑的Ca/钙调蛋白激酶和蛋白激酶C。研究还将着重于确定磷酸化对酶的特性。这些研究将涉及分析 Ca/钙调蛋白激酶磷酸化酶的动力学活化剂蛋白和蛋白激酶的存在和不存在 C磷酸化酶。大鼠脑酶的两种PI形式将分离以确定它们是否代表天然和磷酸酶。对培养细胞的研究将用于呈现体内聊天磷酸化的证据并评估效果乙酰胆碱合成上的磷酸化。隔离和含有活性位点半胱氨酸，精氨酸的肽的测序计划制定组氨酸残留物。这些的识别酶中的残留物将为您提供基础识别活性位点，并将作为站点的焦点特定的诱变。