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.

本申请提出的研究是一项综合调查叶绿体能量转导 H+-ATP 合酶复合物 (CF-o-CF1 复杂的）。本研究分为三个独立但又相互关联的研究一般领域。目标是： 1.（第一部分）物理定位 CF1 上的 ATP 合酶活性位点，对构成活性成分的 CF1 多肽部分进行测序地点。利用碱基和核糖环修饰的腺嘌呤核苷酸光亲和力类似物，维管植物CF1将被共价修饰。亚单位将确定结合类似物的那些。修饰后的亚基将是片段化，共价修饰的肽将被分离并已测序。 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子将从两个物种中分离出来藻类杜氏藻。部分解离和异源后重构后，耦合因子的特性将被确定。他们的亚基结构将在化学和免疫学上。它们的活性将受到巯基定向的调节试剂，并且将确定试剂的结合位点。 3.（第三部分）莱茵衣藻的生物起源研究 CF-o-CF1 复合物。莱茵衣藻 CF1 复合体的非变性亚基将被分离并重组以再生活性复合物。这 CF-o-CF1复合物将被纯化，其亚基组成和化学计量确定。来自 C 突变株的偶联因子。将研究磷酸化缺陷的莱茵哈迪。他们的缺陷将通过检查分离的蛋白质、编码基因来确定对于多肽及其 mRNA。最后，机制将 CF-o 疏水亚基 III 定向插入叶绿体类囊体膜将在重建系统中进行原位检查。