An efficient method for quantitative determination of cellular ATP synthetic activity.
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 to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. 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 treatment was used to make Escherichia coli cells permeable. ATP was 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 activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.
Hara
Kiyotaka Y
KY
Biofrontier Laboratories Kyowa Hakko Kogyo Co. Ltd. 3-6-6 Asahimachi Machidashi, Tokyo 194-8533, Japan.
Mori
Hideo
H
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001419:Bacteria / Q000145:classification / Q000378:metabolism; D002463:Cell Membrane Permeability; D006019:Glycolysis; D049449:Luminescence; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases