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This version was published on April 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 3, 310-317 (2006)
DOI: 10.1177/1087057105285112

An Efficient Method for Quantitative Determination of Cellular ATP Synthetic Activity

Kiyotaka Y. Hara

Hideo Mori

Biofrontier Laboratories Kyowa Hakko Kogyo Co. Ltd. 3-6-6 Asahimachi Machidashi, Tokyo 194-8533, Japan hmori{at}kyowa.co.jp

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult tomeasure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reactionwas used for the quantitativemeasurement. Under the used 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 dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatmentwas used tomake Escherichia coli cells permeable. ATPwas discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activitywas calculated formthe slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.

Key Words: ATP synthesis • glycolysis • luciferin • luciferase • rapid screening


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