Summary
“Permeable Cell Assay” is an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source for biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this assay, bioluminescence from the luciferin–luciferase reaction is used for the quantitative measurement. Under the assay condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure cellular ATP synthetic activity, combination of osmotic shock and detergent, Triton X-100 treatment is used to make bacterial cells permeable. ATP is discharged from permeable cells and reacted with external luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence is increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity is calculated from the slope of linearly increasing bioluminescence.
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Acknowledgments
The author is grateful to Dr. Hirotada Mori and Dr. Tomoya Baba at the Nara Advanced Institute of Science and Technology for providing us BW25113 and atp mutant strains. This study was carried out in Biofrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd. The author would like to thank many supports from the members of this laboratory, especially Dr. Hideo Mori for his management and Natsuka Shimodate for her technical assistance. This work was carried out as a part of The Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by the Ministry of Economy, Trade, & Industry (METI) and was supported by the New Energy and Industrial Technology Development Organization (NEDO).
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Hara, K.Y. (2009). Permeable Cell Assay: A Method for High-Throughput Measurement of Cellular ATP Synthetic Activity. In: Koga, H. (eds) Reverse Chemical Genetics. Methods in Molecular Biology™, vol 577. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-232-2_19
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