ADP

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         MitoPedia: SUIT         MiP and biochemistry         Preprints and history



MitoPedia

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

MitoPedia topics: Substrate and metabolite 


Questions.jpg



Application in HRR

D: ADP (Adenosine 5'diphosphate potassium salt, C10H15N5O10P2K),
Merck 117105-1GM (Calbiochem) FW = 501.3; store at 4 °C. Recommended due to lower ATP impurities, we use 'ADP-Mg' in Oroboros Lab.


Alternative source: Sigma A 5285, 1 g, store at -20 °C; FW = 501.3.
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.


Preparation of 500 mM ADP stock solution (dissolved in H2O):
  1. Weigh 501.3 mg of ADP = 1 mmol ADP
  2. Add 1.2 mL H2O (ADP is not dissolved at this stage).
  3. Neutralize with 5 M KOH (approx. 450 µL). ADP will dissolve after addition of KOH.
  4. Check pH and adjust to 7 with 5 M KOH if necessary.
  5. Adjust final volume to 2 mL and divide into 0.2 mL portions.
  6. Store at -80 °C.


Preparation of 500 mM ADP stock solution with 300 mM free Mg2+ (dissolved in H2O):
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, FW = 203.3)
  1. Weigh 501.3 mg of ADP = 1 mmol ADP.
  2. Add 1.2 mL H2O (ADP is not dissolved at this stage).
  3. Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
  4. Add 121,98 mg MgCl2. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT.
  5. Check pH and adjust to 7 with 5 M KOH if necessary.
  6. Adjust final volume to 2 mL and divide into 0.2 mL portions.
  7. Store at -80 °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):
Merck (former Calbiochem): 117105 (Adenosine 5ʹ-Diphosphate, Potassium Salt), store at -20°C.
  1. Weigh 1.0026 g, dilute in H2O
  2. Adjust pH to 6.9 with KOH, preferably on ice (with pHmeter calibrated on the same condition)
  3. Complete with H2O to 10 mL
  4. Aliquote (200 µL) and store at -20°C.
  5. Avoid thawing and re-freezing the aliquotes.
  6. The concentration can be corrected by measuring the absorbance at 260 nm and using an extinction coefficient factor of εM = 15400 M-1⋅cm-1


O2k manual titrations MiPNet09.12 O2k-Titrations
500 mM ADP:
  • Titration volume: 4-20 µL using a 25 µL syringe (2 mL O2k-chamber).
  • Final concentration: 1-5 mM.
200 mM ADP for experiments with Magnesium Green:
  • Titration volume: 10-50 µL using a 25 µL syringe (2 mL O2k-chamber).
  • Final concentration: 1-5 mM.

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.