MOLECULAR BIOLOGY OF CALCIUM PATHWAYS IN TRYPANOSOMES

锥虫钙途径的分子生物学

• 批准号: 3137727
• 负责人: Lawrence Ruben
• 金额: $ 12.71万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-03-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3137727
• 关键词: Trypanosoma; calcium binding protein; endonuclease; gel filtration chromatography; laboratory mouse; laboratory rabbit; laboratory rat; messenger RNA; molecular biology; oligonucleotides; pentosyltransferase; protein sequence; tritium

Calcium ions are used by all eukaryotic cells as regulatory molecules to coordinate complex and diverse cellular activities. Calcium-binding proteins (CaBPs) have evolved to mediate cellular responsiveness to calcium ions and to maintain low intracellular calcium concentrations. The following proposal is designed to systematically identify and characterize CaBPs in Trypanosoma brucei rhodesiense. Preliminary experiments have identified 8 CaBPs in T. b. rhodesiense. Four of these proteins, including a developmentally regulated CaBP have been purified. The specific goals of this project are to: 1) develop large scale preparative purification protocols for each of the CaBPs identified with emphasis on the four CaBPs that have already been purified on a small scale; 2) determine calcium binding properties of the purified proteins by equilibrium dialysis or by the gel filtration method of Hummel and Dreyer; 3) sequence portions of the purified proteins by automated Edman degredation and determine homologies with any other protein in the Protein Sequence Database of the Protein Identification Resource (1985,V5.0); 4) prepare polyclonal antibodies to selected proteins and use them in fluorescent and colloidal gold immunolocalization studies to identify the site of activity of the different CaBPs; 5) develop a gel overlay procedure to detect other proteins that associate with CaBPs; 6) detect the regulatory CaBP, protein kinase C, if it is present, by phosphorylase activity or as the (20-3H)phorbol 12,13 dibutyrate binding protein; 7) identify genes encoding the different CaBPs by screening an expression library with combinations of the following: i) polyclonal antibodies to different CaBPs, ii) synthetic oligonucleotides homologous with amino acid sequences and iii) 45 Ca using the same 45Ca gel overly procedure originally used to identify CaBPs in cell homogenates and 8) characterize the genes encoding the CaBPs in terms of: i) endonuclease maps, ii) nucleotide sequence, iii) chromosome localization and iv) corresponding mRNAs. Ultimately, this study will characterize major components of calcium-dependent regulatory pathways in African trypanosomes and will likely reveal sensitive processes that, in the long run, can be targeted in the development of trypanocidal therapies.

所有真核细胞都使用钙离子作为调节分子 协调复杂多样的细胞活动。 钙结合 蛋白质 (CaBP) 已进化为介导细胞对钙的反应 离子并维持低细胞内钙浓度。 这 以下提案旨在系统地识别和表征 罗得西亚布氏锥虫中的 CaBP。 初步实验有 在 T. b 中鉴定出 8 个 CaBP。罗德西亚。 其中四种蛋白质，包括 发育调节的 CaBP 已被纯化。 具体目标 该项目的目的是：1）开发大规模制备纯化 针对每个 CaBP 确定的协议，重点是四个 CaBP 已经小规模纯化； 2) 测定钙 通过平衡透析或通过纯化的蛋白质的结合特性 Hummel和Dreyer的凝胶过滤法； 3) 序列部分 通过自动 Edman 降解纯化蛋白质并确定同源性 与该蛋白质的蛋白质序列数据库中的任何其他蛋白质 识别资源（1985，V5.0）； 4) 制备多克隆抗体 选择蛋白质并将其用于荧光金和胶体金 免疫定位研究以确定其活性位点 不同的CaBP； 5) 开发凝胶覆盖程序来检测其他 与 CaBP 相关的蛋白质； 6) 检测调节CaBP、蛋白 激酶 C（如果存在）通过磷酸化酶活性或作为 (20-3H)佛波醇12,13二丁酸结合蛋白； 7) 识别编码基因 通过筛选表达文库来筛选不同的 CaBP 以下：i) 针对不同 CaBP 的多克隆抗体，ii) 合成的 与氨基酸序列同源的寡核苷酸和iii) 45 Ca使用 与最初用于识别 CaBP 的 45Ca 凝胶覆盖程序相同 细胞匀浆和 8) 表征编码 CaBP 的基因 包括：i) 核酸内切酶图谱，ii) 核苷酸序列，iii) 染色体 定位和 iv) 相应的 mRNA。 最终，这项研究将 描述钙依赖性调节途径的主要组成部分 非洲锥虫可能会揭示敏感过程， 从长远来看，可以有针对性地开发杀锥虫疗法。