ADP

high-resolution terminology - matching measurements at high-resolution

ADP

Description

Adenosine diphosphate is a nucleotide. In OXPHOS core metabolism, ADP is a substrate of ANT and ATP synthase in the phosphorylation system. ADP is the discharged or low-energy counterpart of ATP. ADP can accept chemical energy by regaining a phosphate group to become ATP, in substrate-level phosphorylation (in anaerobic catabolism), at the expense of solar energy (in photosynthetic cells) or chemiosmotic energy (respiration in heterotrophic cells). ADP is added to mitochondrial preparations at kinetically saturating concentrations to induce the active state for evaluation of OXPHOS capacity.

Abbreviation: D

Reference: MiPNet03.02, MiPNet09.12

Application in HRR

D: ADP (Adenosine 5'diphosphate potassium salt, C₁₀H₁₅N₅O₁₀P₂K),

Merck 117105-1GM (Calbiochem) M = 501.3 g·mol^-1; store at 4 °C. Recommended due to lower ATP impurities, we use this ADP for preparation of 'ADP-Mg' solution in Oroboros Lab.

Alternative source: Sigma A 5285, 1 g, store at -20 °C; M = 501.3 g·mol^-1.

To keep free [Mg²⁺] constant during respiration measurement in MiR06 or MiR05, mix ADP with MgCl₂ (0.6 mol MgCl₂/mol ADP).

To find out more about the importance of adding Mg²⁺, please refer to here

MgCl₂ (Scharlau MA0036: MgCl₂.6H₂O, M = 203.3 g·mol^-1)

For instructions without MgCl₂ and with smaller amounts of ADP see Talk:ADP

Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.

Preparation of 500 mM ADP stock solution with 300 mM free Mg²⁺ (dissolved in H₂O):

1. Take 1 g of ADP (can be orderd in portions of 1 g per vial).
2. Add 2.3 mL H₂O (ADP is not dissolved at this stage).
3. Neutralize with 5 M KOH (approx. 900 µl). ADP will dissolve after addition of KOH.
4. Add 243.96 mg MgCl₂. White precipitate will occur, which will dissolve during 1-2 min stirring or shaking.
5. Check pH and adjust to 7 with 5 M KOH if necessary (usually about 30 to 50 µl). Addition of KOH causes precipitation. Carefully stir or shake it until the solution is clear again, then add the next droplet of KOH.
6. Adjust final volume to 4 mL and divide into 0.2 mL portions.
7. Store at -20 °C.

Comment: In some cases, a white precipitate was observed after thawing the 'ADP-Mg' solution. If this is the case we recommend to prepare the ADP stock solution and a MgCl₂ solution (0.6 mol MgCl₂/mol ADP) separately and perform a titration of both solutions in immediate succession.

Preparation of 200 mM ADP stock solution without Mg²⁺ for experiments with Magnesium Green (dissolved in H₂O):

» O2k manual titrations MiPNet09.12 O2k-Titrations

500 mM ADP:

• 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.

200 mM ADP for experiments with Magnesium Green:

• Titration volume (2-mL O2k-chamber): 10-50 µL using a 50 µL Hamilton syringe.
• Final concentration: 1-5 mM.

DatLab oxygen flux: performance and data analysis

• Flux increases to OXPHOS capacity at kinetically saturating [ADP]. The full activation of ADP may require time, however for the evaluation of OXPHOS capacity it is needed to reach the steady state.
• Recommendation: use MgADP2+.
• Recommendation: 2.5 mM final conc, 10 µL titration. Information on optimal [ADP]: Gnaiger_2001_Respir_Physiol
• Recommendation for permeabilized fibers: 5 mM or higher final conc. Information on optimal [ADP]: Gnaiger_2001_Respir_Physiol, Permeabilized muscle fibers.
• Guidelines for performing and evaluating respirometric assays: DatLab_oxygen_flux:_performance_and_data_analysis

ADP dependence of respiration

The assumption of linearity (linear regression of oxygen concentration over time) is frequently not valid for various reasons other than oxygen kinetics. In classical ‘State 3’, ADP levels are ‘high’ (Chance and Williams, 1955), but not necessarily saturating (MitoPedia: Respiratory states). Then, an ADP-dependent decline of respiration is observed immediately after titration of a sub-saturating concentration of ADP, which is obscured by any linear regression. This has caused in the past a tremendous underestimation of the apparent Km for ADP, perpetuated even today with the uncritical application of non-adequate software implementing the simple linearity approach only. For a critical approach to ADP kinetics, see Gnaiger et al 2000 and Gnaiger 2001.

» Keywords

Specific

» ANT

» OXPHOS

» OXPHOS capacity

» Phosphorylation system

General

» ATP

» ATP synthase

» MiR06

» Mitochondrial preparations

» O2k-Titrations

References

Bioblast link	Reference	Year
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
BEC 2020.1 doi10.26124bec2020-0001.v1	Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1	2020
Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum	Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum
MiPNet03.02 Chemicals-Media	Selected media and chemicals for respirometry with mitochondrial preparations.	2016-08-30
Vajda 2009 FEBS J	Vajda S, Mándi M, Konràd C, Kiss G, Ambrus A, Adam-Vizi V, Chinopoulos C (2009) A re-evaluation of the role of matrix acidification in uncoupler-induced Ca2+ release from mitochondria. FEBS J 276:2713-24.	2009
MiPNet09.12 O2k-Titrations	O2k manual titrations: SUIT protocols with mitochondrial preparations.	2020-08-17

MitoPedia topics: Substrate and metabolite