Study on Mechanisms of ATP Synthesis in Single Mitochondria

单线粒体ATP合成机制研究

• 批准号: 10680627
• 负责人: OHTA Yoshihiro
• 金额: $ 1.92万
• 依托单位: Tokyo University of Agriculture and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680627/
• 关键词: mitochondria; ATP; fluorescence microscopy; imaging; permeability transition; membrane potential; NADH; flavin; 蛍光画像; ATP合成; フラビン蛋白質

The mitochondrial activities related to ATP production were measured in single mitochondria with fluorescence microscopy. So far, studies on mitochondria have been performed with mitochondrial suspension. Therefore, it has been impossible to detect asynchronous phenomena like the opening and the closing of a channel. In the present study, we developed the technique to measure the activity of single mitochondria to detect the phenomena that has not been detected with mitochondria suspension.To observe single mitochondria with fluorescence microscopy, mitochondria were adsorbed on the cover slip coated with polyphenolic protein. Since the adsorption did not decrease the ATP production rate in mitochondria, this method for adsorption was elucidated to be useful for the measurements of mitochondrial activity. Upon addition of malate as a respiratory substrate, the time-courses of NADH formation and Flavin reduction in single mitochondria was similar to the results obtained for mitochondrial suspension. On the other hand, mitochondria showed the generation and disruption of the membrane potential due to the opening and the closing of the permeability transition pore. The opening and the closing depends on the redox state of the electron transport chain.

用荧光显微镜在单线粒体中测量与ATP产生相关的线粒体活性。到目前为止，已经通过线粒体悬浮液进行了有关线粒体的研究。因此，不可能检测出异步现象，例如通道的开口和关闭。在本研究中，我们开发了一种测量单线粒体活性的技术，以检测尚未用线粒体悬浮液检测到的现象。为了观察带有荧光显微镜的单线粒体，线粒体在用多酚蛋白的覆盖层上吸附在覆盖物上。由于吸附并不能降低线粒体中的ATP产生速率，因此阐明了这种吸附方法可用于测量线粒体活性的测量。添加苹果酸作为呼吸底物后，单线粒体中NADH形成和黄素还原的时光与线粒体悬浮液的结果相似。另一方面，线粒体显示出由于渗透率过渡孔的开口和关闭而产生的膜电位的产生和破坏。开口和关闭取决于电子传输链的氧化还原状态。

项目成果

Hoolahan et al.: "Molecular Steroidogenesis"Calcium Ion Enhances the Production of NAD(P)H and Pregnenolone in Bovine Adrehocortical Mitochondria. 442 (2000)

Hoolahan 等人：“分子类固醇生成”钙离子增强牛肾上腺皮质线粒体中 NAD(P)H 和孕烯醇酮的产生。

渡辺功一他: "蛍光顕微鏡による単離ミトコンドリアの観察方法"生物物理. 224. 258-260 (1999)

Koichi Watanabe 等人：“使用荧光显微镜观察分离线粒体的方法”生物物理学 224. 258-260 (1999)

Yamazaki,T.: "Calcium Ion as a Second Messenger for O-Nitrophenylsulfenyl-Adrenocorticotropin (ACTH) and ACTH in Bovine Adrenal Steroidogenesis" Endocrinology. 139. 4765-4771 (1998)

Yamazaki,T.：“钙离子作为 O-硝基苯硫基促肾上腺皮质激素 (ACTH) 和 ACTH 在牛肾上腺类固醇生成中的第二信使”内分泌学。

Suzuki-Tomii et al.: "Comparative Experiments of Protein Denaturation for Studying Structural Difference of Homologous Membrane Proteins : P450scc and P450_<11β>"Jpn.J.Appl.Phys.. 38. 298-301 (1999)

Suzuki-Tomii 等：“用于研究同源膜蛋白结构差异的蛋白质变性的比较实验：P450scc 和 P450_<11β>”Jpn.J.Appl.Phys.. 38. 298-301 (1999)

Makoto Yamada: "Dynamic mobility of genetically expressed fusion protein between cytochrome P450IAI and NADPH-cytochrome P450 reductase in yeast microsomes"Biochemistry. 38. 9465-9470 (1999)

Makoto Yamada：“酵母微粒体中细胞色素 P450IAI 和 NADPH-细胞色素 P450 还原酶之间遗传表达的融合蛋白的动态迁移率”生物化学。