Adenosine triphosphate is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate (ADP).
Reference: MiPNet03.02 Chemicals-Media
Application in HRR
- T: ATP (Adenosine 5'-triphosphate disodium salt hydrate; C10H14N5O13P3Na2* xH2O), Sigma-Aldrich: A2383, store at -20 °C, CAS: 34369-07-8, M = 551.1 g·mol-1
- Alternative source: Merck 1191-1GM (Calbiochem, Adenosine 5′-Triphosphate Disodium Salt; M = 551.2 g·mol-1; store at -20 °C).
- Preparation of 500 mM ATP stock solution
- Weigh 551.1 mg of ATP (Manhydrous basis = 551.1 g·mol-1).
- Add 1.2 mL H2O.
- Neutralize with 5 M KOH (approx. 400 µL) - ATP dissolves after addition of KOH (this is Solution A).
- Check pH and adjust to pH 7 if necessary.
- Adjust final volume to 2 mL and divide into 0.2 mL portions.
- Store at -20 °C (-80 °C should be preferred for long-time storage).
- Preparation of 500 mM ATP stock solution with 400 mM free Mg2+
- MgCl2 (Scharlau MA0036: MgCl2.6H2O, M = 203.3 g·mol-1)
- Add 162.64 mg MgCl2 to Solution A. A white precipitate forms, which dissolves after 1-2 min of stirring on magnetic stirrer at room temperature.
- Follow steps 4-6 of the above instructions.
- Comment: In some cases it was observed, that after defreezing of the 'ATP-Mg' a white precipitate forms again. If this is the case we recommend to prepare the 500 mM ATP stock solution and a MgCl2 solution (0.8 mol MgCl2 /mol ATP) seperately and perform a titration of both solutions in series.
- » O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 4-20 µL using a 25 µL Hamilton syringe.
- Titration volume (0.5-mL O2k-chamber): 1-5 µL using a 10 µL Hamilton syringe.
- Final concentration: 1-5 mM.
- ATP may be titrated to mt-preparations in ET-pathway competent substrate states to quantify ATPase activity before addition of ADP (D). As ATP is hydrolysed to ADP, OXPHOS is activated by ADP and respiration increases accordingly. High-quality isolated mitochondria contain zero or very low ATPase activity, such that resipration in the absence and presence of ATP is identical.
- Intracellular ATP levels are in the mM range, but many respiratory studies of mt-preparations are carried out at zero or µM concentrations of ATP. ADP kinetics of isolated rat liver mitochondria reveals a K´m,D of 56 µM ADP in the presence of 2 mM ADP (Gnaiger 2001), which is higher than the frequently cited value of 15 to 20 µM ADP.
- ATP stabilizes mitochondrial function and is therefore used in preservation solutions BIOPS and MiP03 (MiPNet03.02 Chemicals-Media). This may provide a rationale to add mM concentrations of ATP in the LEAK state in the absence of ATPase activity (Gnaiger et al 2000). In permeabilized fibres with high ATPase activity, ATP might be added after stimulation of respiration by ADP in various SUIT protocols. Arguments against additions of ATP, however, need to be considered, e.g. additional side effects exerted on other sensors in O2k-MultiSensor applications (TPP+).
--Gnaiger Erich 14:20, 16 February 2014 (CET)
Measurement of mitochondrial ATP production
- A fluorimetric technique is available to measure mitochondrial ATP production (or exchange of ADP/ATP by ANT) combined with the O2k, using the O2k-Fluo LED2-Module or O2k-Fluo Smart-Module and the dye Magnesium Green.
- » Magnesium Green
- » Chinopoulos 2014 Methods Enzymol
|Chinopoulos 2014 Methods Enzymol||Chinopoulos C, Kiss G, Kawamata H, Starkov AA (2014) Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption. Methods Enzymol 542:333-48. doi:10.1016/B978-0-12-416618-9.00017-0||2014|
|Davis 1980 Eur J Biochem||Davis EJ, Spydevold O, Bremer J (1980) Pyruvate carboxylase and propionyl-CoA carboxylase as anaplerotic enzymes in skeletal muscle mitochondria. Eur J Biochem 110:255-62.||1980|
|Gnaiger 2001 Respir Physiol||Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. https://doi.org/10.1016/S0034-5687(01)00307-3||2001|
|BEC 2020.1 doi10.26124bec2020-0001.v1||Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. https://doi.org/10.26124/bec:2020-0001.v1||2020|
|Gnaiger 2000 Proc Natl Acad Sci U S A||Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. https://doi.org/10.1073/pnas.97.20.11080||2000|
|MiPNet03.02 Chemicals-Media||Selected media and chemicals for respirometry with mitochondrial preparations.||2016-08-30|
|Taroni 1987 Biochim Biophys Acta||Taroni F, Gellera C, Di Donato S (1987) Evidence for two distinct mitochondrial malic enzymes in human skeletal muscle: Purification and properties of the NAD(P)+-dependent enzyme. Biochim Biophys Acta 916: 446-454.||1987|
|Tuena 1969 Eur J Biochem||Tuena M, Gómez-Puyou A, Peña A, Chávez E, Sandoval F (1969) Effect of ATP on the oxidation of succinate in rat brain mitochondria. Eur J Biochem 11:283-90.||1969|
MitoPedia topics: Substrate and metabolite