✍ Scribed by Peter Ronner; Edward Friel; Katharina Czerniawski; Stefanie Fränkle
No coin nor oath required. For personal study only.
We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-l samples in 0.5-ml microcentrifuge tubes and subsequent 5-l additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon-and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.
📜 SIMILAR VOLUMES
## Abstract Based on experimentally determined concentrations of PCr, __P__~i~, ATP, and H^+^, the ADP and AMP concentrations and the free energy for the ATP hydrolysis in normal human brain and muscle were calculated assuming that the creatine kinase‐ and the myokinase‐catalyzed reactions were in
Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack