Study on the regulatory region of the chloroplast ATP synthase by using peptide antibody

肽抗体研究叶绿体ATP合酶调控区

• 批准号: 11640643
• 负责人: HISABORI Toru
• 金额: $ 2.3万
• 依托单位: Chemical Resources Laboratory, Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640643/
• 关键词: ATP synthase; chloroplast; gamma subunit; thioredoxin; regulation; CF_1; ジスルフィド結合; 調節

To clarify the regulatory mechanism of the chloroplast ATP synthase, we carried out three different approach as follows ; (1) the mutation analysis of the regulatory region of the γ subunit, (2) the investigation of the interaction between thioredoxin and the regulatory region of the γ subunit, (3) the investigation of the interaction between thioredoxin and the chloroplast ATP synthase F_1 part.We first investigated the change of the regulation by the introduction of the mutation at around the regulatory region including two cysteines on the γ subunit. From this study, we concluded that the conformational change of the regulatory region itself is the most critical for the regulation of the enzyme activity. Furthermore, we found that the deletion of the negative charged cluster in the regulatory region results the inversion of the regulation.Secondary, we prepared the mutant thioredoxin to investigate the interaction with ATP synthase. From this study, we could clarify that the chloroplast thioredoxin can induce the important conformational change of the γ subunit to facilitate the reduction of the regulatory disulfide bond only by the protein-protein interaction. Although we prepared the antibody against the active region of the chloroplast thiredoxin to investigate the interaction between thioredoxin and 2ATP synthase, the study on this direction is not successful yet.

为了阐明叶绿体 ATP 合成酶的调节机制，我们进行了以下三种不同的方法； (1)γ亚基调节区的突变分析，(2)硫氧还蛋白与γ亚基调节区之间相互作用的研究，(3)硫氧还蛋白与叶绿体ATP合酶F_1部分之间相互作用的研究。我们首先通过在调节区周围引入突变(包括γ上的两个半胱氨酸)来研究调节的变化。 亚基。从这项研究中，我们得出结论，调节区本身的构象变化对于酶活性的调节是最关键的。此外，我们发现调控区负电荷簇的缺失会导致调控反转。 其次，我们制备了突变体硫氧还蛋白来研究与ATP合酶的相互作用。从这项研究中，我们可以阐明叶绿体硫氧还蛋白只能通过蛋白质-蛋白质相互作用诱导γ亚基的重要构象变化，以促进调节性二硫键的还原。尽管我们制备了针对叶绿体硫氧还蛋白活性区的抗体来研究硫氧还蛋白与2ATP合酶之间的相互作用，但该方向的研究尚未成功。

项目成果

Bald D.: "ATPase activity of a highly stable α_3β_3γ Subcomplex of thermophilic F1 can be regulated by the introduced regulatory region of γ-subunit of chloroplast F1"J.Biol.Chem.. (in press). (2000)

Bald D.：“嗜热 F1 的高度稳定的 α_3β_3γ 亚复合物的 ATP 酶活性可以通过叶绿体 F1 的 γ 亚基的引入调节区域进行调节”J.Biol.Chem..（出版中）。

Stumpp,M.T.,Motohashi,K.,Hisabori,T.: "Chloroplast thioredoxin mutants without active site cysteines facilitate the reduction of the regulatory disulfide bridge on the γ subunit of chloroplast ATP synthase"Biochem.J.. 341. 157-163 (1999)

Stumpp, M.T.、Motohashi, K.、Hisabori, T.：“没有活性位点半胱氨酸的叶绿体硫氧还蛋白突变体促进叶绿体 ATP 合酶 γ 亚基上调节二硫键的还原”Biochem.J. 341. 157-163（ 1999）

Stumpp M.T.: "Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase"Biochmical Journal. 341. 157-163 (1999)

Stumpp M.T.：“没有活性位点半胱氨酸的叶绿体硫氧还蛋白突变体有助于减少叶绿体 ATP 合酶 γ 亚基上的调节二硫键”生物化学杂志 341. 157-163 (1999)。

Hisabori T,Kondoh A,Yoshida M: "The γ subunit in chloroplast F_1-ATPase can rotate in a unidirectional and counter-clockwise manner"FEBS Lett.. 463. 35-38 (1999)

Hisabori T、Kondoh A、Yoshida M：“叶绿体 F_1-ATP 酶中的 γ 亚基可以单向和逆时针方式旋转” FEBS Lett.. 463. 35-38 (1999)

Bald,D.: "ATPase activity of a highly stable α_3β_3γ subcomplex of thermophilic F_1 can be regulated by the introduced regulatory region of γ subunit of chloroplast F_1"Journal of Biological Chemistry. 275. 12757-12762 (2000)

Bald，D.：“嗜热 F_1 的高度稳定的 α_3β_3γ 亚复合物的 ATP 酶活性可以通过叶绿体 F_1 的 γ 亚基的引入调节区域进行调节”《生物化学杂志》275. 12757-12762 (2000)。