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Studies on structure and function of connection.

连接结构和功能的研究。

基本信息

批准号：
02640522
负责人：
SHIN Masateru
金额：
$ 1.02万
依托单位：
Kobe University, Faculty of Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

An assay system for physiological function of connectein, a constituent of the large form of ferredoxin-NADP reductase(FNR-L), was established by modifying the reconstituted NADP photoreducing system originally designed for demonstrating physiological function of FNR-L. FNR-L was highly purified by an improved method and depleted thylakoids were prepared by brief extraction of FNR-L with a detergent, CHAPS. Using these components, NADP photoreducing system was successfully reconstituted with good reproducibility. NADP photoreducing activity of thylakoids was decreased when FNR-L was removed by CHAPS treatment and the decreased activity was fully restored by rebinding of FNR-L. In contrast to FNR-L, monomer FNR, which does not associated with connection, was almost ineffective in restoring the NADPphotoreducing activity of FNR-L-depleted thylakoids. The clear defference in restoration of the NADP photoreducing activity observed between FNR with and without connection reflects functional contribution of connection to the NADP photoreducing reaction of thylakoids. Thus, physiological activity of connection could be quantified by this reconstitution experiment.An attempt was made to analyze an amino acid composition and amino-terminal structure of connectein, since the unique function of the protein may originate from its structure. Its amino-terminal structure was identified up to the 16th residue but showed no sequence homology with any of the polypeptides sequenced so far.

连接蛋白（connectein）是铁氧化还原蛋白-NADP还原酶（FNR-L）的组成成分，通过对原设计用于表征FNR-L生理功能的NADP光还原系统进行修饰，建立了连接蛋白生理功能测定系统。通过改进的方法对FNR-L进行了高纯度纯化，并用CHAPS洗涤剂对FNR-L进行了短时间萃取，制备了贫类囊体。利用这些成分成功地重建了NADP光还原体系，重现性好。当CHAPS处理去除FNR-L后，类囊体的NADP光还原活性降低，FNR-L的再结合使其完全恢复。与FNR- l相比，与连接无关的单体FNR在恢复FNR- l耗尽的类囊体的nad光还原活性方面几乎无效。有连接和无连接的FNR在恢复NADP光还原活性方面的明显差异反映了连接对类囊体NADP光还原反应的功能贡献。因此，连接的生理活动可以通过这个重建实验来量化。由于连接蛋白的独特功能可能来源于其结构，因此对其氨基酸组成和氨基末端结构进行了分析。其氨基末端结构在第16个残基前已被鉴定，但未与任何已测序的多肽序列同源。