CHOLINE ACETYLTRANSFERASE

胆碱乙酰转移酶

• 批准号: 2683111
• 负责人: Louis B. Hersh
• 金额: $ 25.11万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-08 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2683111
• 关键词: X ray crystallography; active sites; choline acetyltransferase; complementary DNA; enzyme activity; enzyme induction /repression; gene mutation; genetic promoter element; genetically modified animals; human genetic material tag; laboratory rat; molecular cloning; nucleic acid sequence; phosphorylation; posttranslational modifications; protein sequence; recombinant proteins; site directed mutagenesis; tissue /cell culture; transcription factor; X ray crystallography; active sites; choline acetyltransferase; complementary DNA; enzyme activity; enzyme induction /repression; gene mutation; genetic promoter element; genetically modified animals; human genetic material tag; laboratory rat; molecular cloning; nucleic acid sequence; phosphorylation; posttranslational modifications; protein sequence; recombinant proteins; site directed mutagenesis; tissue /cell culture; transcription factor

The over-all objective of this project is to understand the regulation of the enzyme choline acetyltransferase through both transcriptional and post translational mechanisms. The project involves 5 specific aims. These include the elucidation of the three dimensional structure of the enzyme by x-ray defraction analysis. Crystals of the rat enzyme which defract to 4.5 angstroms have been obtained and will be used for initial structural determination. Crystalization conditions will be optimized to obtain crystals which defract to a higher resolution. Studies on the regulation of the enzyme by post-translational modification will involve an analysis of the use of alternative translational start sites to produce two molecular forms of human ChAT and the potential for one of these enzyme forms to serve as a precursor for the membrane bound form of the enzyme. Regulation of the enzyme by phosphorylation is implicated by the finding that phosphorylated recombinant rat ChAT derived from expression in plant cells exhibits 1/20 the activity of non-phosphorylated recombinant rat ChAT expressed in E. coli. Studies will be conducted on the in vitro phosphorylation of the enzyme and the effect of phosphorylation on activity. The phosphorylation state of the enzyme derived from rat brain will be assessed. Site directed mutagenesis will be used to assess the role of specific arginine, histidine, and cysteine residues in catalysis. The regulation of the enzyme at the transcriptional level will be investigated by studying those elements in the human gene which define cholinergic specific expression. Putative regulatory regions of the gene will be studied by a combination of cell transfection studies and the analysis of transgenic mice. Lastly the transcription factors which bind to the regulatory sequences in the gene will be characterized by gel shift and footprint analysis. The former technique used to isolate and characterize any unique transcription factors whose function will be studied.

该项目的所有目标是了解 酶胆碱乙酰基转移酶通过转录和邮政 翻译机制。该项目涉及5个具体目标。这些 包括阐明酶的三维结构 通过X射线剥落分析。大鼠酶的晶体碎裂 4.5埃已获得，并将用于初始结构 决心。将优化结晶条件以获得 降解为更高分辨率的晶体。调节的研究 通过翻译后修饰的酶将涉及分析 使用替代翻译起始站点生产两个 人类聊天的分子形式以及这些酶之一的潜力 作为酶的膜结合形式的前体形式。 通过磷酸化调节酶的调节与发现有关 从植物中表达得出的磷酸化重组大鼠聊天 细胞表现出1/20的非磷酸化重组大鼠的活性 聊天在大肠杆菌中表达。研究将在体外进行 酶的磷酸化和磷酸化对 活动。源自大鼠脑的酶的磷酸化状态 将被评估。定向诱变将用于评估 特异性精氨酸，组氨酸和半胱氨酸残基在催化中的作用。 转录水平的酶的调节将是 通过研究人类基因中的这些元素来调查 胆碱能特异性表达。基因的推定调节区域 将通过细胞转染研究和 转基因小鼠的分析。最后是结合的转录因子 基因中的调节序列将以凝胶移位为特征 和足迹分析。以前的技术用于隔离和 表征其功能将是的任何独特的转录因子 研究。