THE THYLAKOID ENERGY TRANSDUCING ATPASE COMPLEX

类囊体能量转导ATP酶复合物

基本信息

批准号：
3279375
负责人：
BRUCE R SELMAN
金额：
$ 16.01万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-08-01 至 1992-07-31
项目状态：
已结题

项目摘要

The research proposed in this application is a comprehensive investigation of the chloroplast energy transducing H+-ATP synthase complex (CF-o-CF1 complex). This study is subdivided into three independent but interrelated general areas. The goals are: 1. (Part I) To physically locate the ATP synthase active site on CF1 and to sequence those sections of the CF1 polypeptides that make up the active site. Using base and ribose ring modified adenine nucleotide photoaffinity analogs, the vascular plant CF1 will be covalently modified. The subunits that bind the analogs will be determined. Modified subunits will be fragmented, and the covalently modified peptides will be isolated and sequenced. 2. (Part II) To study subunit-subunit interactions of CF1. Coupling factors with different properties will be isolated from two species of the alga Dunaliella. After partial dissociation and heterologous reconstitution, the properties of the coupling factors will be determined. Their subunit structures will be compared both chemically and immunologically. Their activities will be modulated by sulfhydryl-directed reagents, and the binding sites for the reagents will be determined. 3. (Part III) To study the biogenesis of the Chlamydomonas reinhardi CF-o-CF1 complex. Non-denatured subunits of the C. reinhardi CF1 complex will be isolated and reconstituted to regenerate an active complex. The CF-o-CF1 complex will be purified and its subunit composition and stoichiometry determined. Coupling factors from mutant strains of C. reinhardi, defective in phosphorylation, will be studied. Their defects will be established by examining the isolated proteins, the genes coding for the polypeptides, and their mRNAs. Finally, the mechanism for the directed insertion of the CF-o hydrophobic subunit III into the chloroplast thylakoid membrane will be examined in situ in a reconstituted system.

本应用程序中提出的研究是一项全面的调查叶绿体能量转导H+-ATP合酶复合物（CF-O-CF1）复杂的）。这项研究细分为三个独立但相互关联一般区域。目标是： 1。（第I部分）在CF1和为构成活性的CF1多肽的那些部分地点。使用碱和核糖环修饰的腺苷核苷酸光附近类似物，血管植物CF1将被共价修改。亚基结合类似物将被确定。修改的亚基将是碎片，将分离的经过重价修饰的肽被隔离，并测序。 2。（第二部分）研究CF1的亚基 - 亚基相互作用。耦合具有不同特性的因素将从两种物种中分离出来藻类Dunaliella。部分解离和异源重建，将确定耦合因子的特性。它们的亚基结构将通过化学和免疫学。他们的活动将由硫磺指导调节试剂将确定试剂的结合位点。 3。（第三部分）研究衣原体的生物发生 CF-O-CF1复合物。 C. reinhardi CF1复合物的非定义亚基将被隔离并重组以再生活跃的复合物。这 CF-O-CF1复合物将被纯化及其亚基组成和确定化学计量。来自C的突变菌株的耦合因子。 Reinhardi（磷酸化缺陷）将被研究。他们的缺陷将通过检查孤立蛋白（基因编码）来建立用于多肽及其mRNA。最后，将CF-O疏水亚基III的定向插入到叶绿体中类囊体膜将在重构系统中进行原位检查。