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Utilizing ATeam Fluorescent Proteins for Assessing ATP Synthesis and Hydrolysis In Vitro and in Isolated Yeast Mitochondriaтезисы доклада Тезисы
- Авторы: Lapashina Anna S., Tretyakov Danila O., Galkina Kseniia V., Markova Olga V., Knorre Dmitry A., Feniouk Boris A.
- Сборник: Biochimica et Biophysica Acta (BBA) - Bioenergetics
- Серия: Supplement 2024 EBEC abstracts
- Том: 1865
- Тезисы
- Год издания: 2024
- Издательство: Elsevier BV
- Местоположение издательства: Netherlands
- Номер статьи: 149308
- DOI: 10.1016/j.bbabio.2024.149308
- Аннотация: ATeam fluorescent proteins are extensively utilized to monitor ATP concentrations within living cells due to their ability to reversibly bind ATP. This binding induces a conformational change, detectable through fluorometry. We purified ATeam from bacteria and used it for in vitro measurements of ATPase activity of ATP-synthases from Escherichia coli, Bacillus subtilis, and thermophilic Bacillus sp. PS3 under conditions when ADP and phosphate are present. Traditional methods of ATPase activity measurements based on registration of phosphate release, on monitoring the acidification of the medium, or on regeneration of ADP to ATP coupled to NADH oxidation do not allow real-time measurements under such conditions. We found that the ATPase activity of Escherichia coli enzyme was significantly suppressed upon addition of ADP and phosphate. However, the enzymes from Bacillus subtilis and Bacillus sp. PS3 retained most of their ATPase activity. It was also found that the level of ADPinhibition of Bacillus subtilis ATP synthase decreases with increasing temperature. When expressed with a mitochondrial address in yeast cells, ATeam is accumulated in mitochondria. We purified mitochondria with ATeam and measured ATP synthesis and hydrolysis following the spectral changes of ATeam fluorescence. In conclusion, ATeam proteins offer a practical and effective method for real-time analysis of ATP synthesis and hydrolysis both in vitro and in isolated mitochondria. This study was funded by the Russian Scientific Foundation (Grant No. 20-14-00268).
- Добавил в систему: Лапашина Анна Сергеевна