All ATP producing and ATP degrading enzymatic reactions can be continuously monitored by measuring the light from firefly luciferase/luciferin being present in the reaction mixture. Examples include protein kinases67, adenylate kinase14, pyruvate kinase6, pyruvate phosphate dikinase, glycerol kinase43, 44, creatine kinase isoenzymes8, 10, 18, 22, ATPases14, ATP synthetases49, aminoacyl tRNA synthetase. Substrates to such enzymes can be measured kinetically or with endpoint assays. Examples include ADP, AMP, pyrophosphate. When continuously monitoring ATP the luciferase ATP depletion should be negligible compared to the enzymatic reactions being measured. ATP concentrations from 1 to 1,000,000 pmol/L can be monitored. ATP depletion assays are best set up as 1st order reactions plotting the natural logarithm of the light versus time. The slope in such plots is the rate constant of the reaction. ATP forming reactions can be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test.
- Monitoring of ATP forming and ATP degrading enzymatic reactions.
- Assays of enzymes and metabolites that can be coupled to ATP formation or degradation.
- Monitoring of cell lysis (ATP or ATP producing enzymes)
- Monitoring of oxidative phosphorylation
- Monitoring of photophosphorylation
ATP Kit SL, Kinase RR Kit, ATP Standards
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Lundin, A. (2000) Use of Firefly Luciferase in ATP-Related Assays of Biomass, Enzymes, and Metabolites. Methods in Enzymology, 305, 346-370.