Permeable cell assay: a method for high-throughput measurement of cellular ATP synthetic activity.
"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.
Hara
Kiyotaka Y
KY
Consolidated Research Institute for Advanced Science and Medical Science and Medical Care, Waseda University, Tokyo, Japan.
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D025243:Bacterial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D057166:High-Throughput Screening Assays / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D009154:Mutation; D017830:Octoxynol; D009997:Osmotic Pressure
D000255:Adenosine Triphosphate; D017830:Octoxynol; D025243:Bacterial Proton-Translocating ATPases