Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity.
Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity.
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DOI：
10.1186/s12934-015-0239-z
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发表时间：
2015-04-16
影响因子：
6.4
通讯作者：
Kondo A
中科院分区：
文献类型：
--
作者：
Ye X;Morikawa K;Ho SH;Araki M;Nishida K;Hasunuma T;Hara KY;Kondo A
线粒体ATP合成的测量是比较细胞能量性能的关键方法。然而，线粒体的分级分离需要大量的细胞，冗长的纯化程序，以及避免损伤完整线粒体的极端谨慎，使其成为线粒体功能高通量研究的最高障碍。为了评估酿酒酵母中参与氧化磷酸化的45个基因，我们旨在发展一种简单快速的方法来测量线粒体ATP合成。为了获得功能性线粒体，S.用酵母菌酶裂解酿酒酵母细胞，然后进行两步，低速然后高速离心。使用萤火虫荧光素酶测定，测定线粒体的ATP合成活性。在线粒体抑制剂存在下ATP合成的减少证实了分离的粗线粒体的功能。线粒体ATP合成相关蛋白基因的缺失表明，线粒体ATP合成相关蛋白依赖于氧化磷酸化。啤酒。与常规方法相比，该方法对S。酿酒酵母线粒体ATP高通量合成活性（MASH方法）简单，需要少量细胞，适合高通量分析。据我们所知，这是第一个报告的快速纯化过程中酵母线粒体适合高通量筛选。
Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low- then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae. Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.
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影响因子：
4.2
作者：
Han, Yong Hwan;Kim, Suhn Hee;Park, Woo Hyun
通讯作者：
Park, Woo Hyun
影响因子：
14.8
作者：
Frezza, Christian;Cipolat, Sara;Scorrano, Luca
通讯作者：
Scorrano, Luca
DOI：
10.1111/j.1432-1033.1987.tb10741.x
发表时间：
1987-02-16
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
OUDSHOORN, P;VANSTEEG, H;GRIVELL, LA
通讯作者：
GRIVELL, LA
影响因子：
3
作者：
Yamamoto, T;Terauchi, S;Shinohara, Y
通讯作者：
Shinohara, Y
DOI：
10.1016/j.bbrc.2010.09.089
发表时间：
2010-10-29
影响因子：
3.1
作者：
Fujikawa, Makoto;Yoshida, Masasuke
通讯作者：
Yoshida, Masasuke