MOLECULAR BIOLOGY OF CALCIUM PATHWAYS IN TRYPANOSOMES

锥虫钙途径的分子生物学

• 批准号: 3137728
• 负责人: Lawrence Ruben
• 金额: $ 13.26万
• 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 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3137728
• 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.

所有真核细胞都将钙离子用作调节分子 协调复杂和多样化的细胞活性。 钙结合 蛋白质（CABP）已演变为介导细胞对钙的反应 离子并保持低细胞内钙浓度。 这 遵循建议旨在系统地识别和表征 Brucei Rhodesiense中的锥虫中的凸轮。 初步实验具有 在T. b中确定了8个驾驶室。罗得教。 这些蛋白质中有四种，包括 受发展监管的CABP已被纯化。 的具体目标 该项目是：1）开发大规模准备纯化 每个CABP的协议，重点是四个Cabps 已经在小规模上纯化了； 2）确定钙 通过平衡透析或通过 Hummel和Dreyer的凝胶过滤方法； 3）序列部分 通过自动化的Edman降解并确定同源性纯化蛋白质 蛋白质的蛋白质序列数据库中的任何其他蛋白质 身份资源（1985，v5.0）； 4）准备多克隆抗体 选择蛋白质并将其用于荧光和胶体金 免疫定位研究以识别 不同的驾驶室； 5）制定凝胶覆盖过程以检测其他 与Cabps关联的蛋白质； 6）检测调节的CABP蛋白质 激酶C，如果存在，则通过磷酸化酶活性或作为 （20-3h）佛罗宝贝12,13二丁酸酯结合蛋白； 7）识别编码的基因 通过筛选表达式库的组合 以下内容：i）与不同CABP的多克隆抗体，ii）合成 与氨基酸序列同源的寡核苷酸，使用III）45 Ca使用 相同的45CA凝胶过度的过程最初用于识别Cabps 细胞匀浆和8）表征编码CABP的基因 ：i）核酸内切酶图，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 表征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 长期可以针对锥虫疗法的发展。