Studies on structure and function of connection.

通过改造重组NADP光还原系统，建立了一种用于检测大型铁氧还蛋白-NADP还原酶（FNR-L）的连接蛋白生理功能的检测系统。采用改进的方法对FNR-L进行了高纯度纯化，并通过洗涤剂CHAPS对FNR-L进行简单提取，制备了贫类囊体。利用这些组分成功地重建了NADP光还原体系，重现性好。当通过CHAPS处理去除FNR-L时，类囊体的NADP光还原活性降低，并且通过FNR-L的重新结合完全恢复降低的活性。与FNR-L相反，与连接无关的单体FNR在恢复FNR-L缺失的类囊体的NADP光还原活性方面几乎无效。在有连接和无连接的FNR之间观察到的NADP光还原活性恢复的明显差异反映了连接对类囊体的NADP光还原反应的功能贡献。因此，连接的生理活性可以通过该重构实验来量化。由于连接蛋白的独特功能可能源于其结构，因此尝试分析连接蛋白的氨基酸组成和氨基末端结构。它的氨基末端结构被确定为第16个残基，但没有显示出与迄今为止测序的任何多肽的序列同源性。

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.