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Difference between revisions of "ADP"

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|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]].
|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]].
|info=[[MiPNet03.02]], [[MiPNet09.12]]
|info=[[MiPNet03.02]], [[MiPNet09.12]]
}}
__TOC__
__TOC__


== Application in [[HRR]] ==
== Application in [[HRR]] ==
{{Chemical_description
|abbr=D
|trivial name=ADP
|complete name=Adenosine 5-diphosphate potassium salt
|chem formula=C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K
|molar mass=501.3
|vendor=Merck (Calbiochem)
|product number=117105-1GM
|store at=4 Ā°C
|sensitivity=
|cas=72696-48-1
|h statements=
|h info=
}}<!--::: '''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K),
::: MerckĀ  117105-1GM ('''Calbiochem''') M = 501.3 gĀ·mol<sup>-1</sup>; store at 4 Ā°C.-->
:::: Recommended due to lower ATP impurities, we use this (Merck-Calbiochem) ADP for preparation of 'ADP-Mg' solutionĀ  in [[AT_Innsbruck_Oroboros |Oroboros Lab]].
:::: Alternative source:Ā  Sigma A 5285, 1 g, store at -20 Ā°C; M = 501.3 gĀ·mol<sup>-1</sup>.


::: '''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K),
:::: ToĀ  keep freeĀ  [Mg<sup>2+</sup>] constant during respirationĀ  measurementĀ  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 molĀ  MgCl<sub>2</sub>/mol ADP).
::: MerckĀ  117105-1GM ('''Calbiochem''') FW = 501.3; store at 4 Ā°C. Recommended due to lower ATP impurities, we use this ADP for preparation of 'ADP-Mg' solutionĀ  in [[AT_Innsbruck_Oroboros |Oroboros Lab]].
:::: To find out more about the importance of adding Mg<sup>2+</sup>, please refer to [https://en.wikipedia.org/wiki/Magnesium_in_biology#Essential_role_in_the_biological_activity_of_ATP here]
:::: '''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, M = 203.3 gĀ·mol<sup>-1</sup>)
:::: For instructions without MgCl<sub>2</sub> and with smaller amounts of ADP see [[Talk:ADP]]


::: Alternative source:Ā  Sigma A 5285, 1 g, store at -20 Ā°C; FW = 501.3.
<!--
Ā 
:::: '''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O):
:::: <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
Ā 
Ā 
::: '''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O):


::::# Weigh 501.3 mg of ADP = 1 mmol ADP
::::# Weigh 501.3 mg of ADP = 1 mmol ADP
Line 23: Line 39:
::::# Adjust final volume to 2 mL and divide into 0.2 mL portions.
::::# Adjust final volume to 2 mL and divide into 0.2 mL portions.
::::# Store at -80 Ā°C.
::::# Store at -80 Ā°C.
-->


:::: '''PreparationĀ  of 500 mM ADP stock solution with 300 mM freeĀ  Mg<sup>2+</sup>''' (dissolved inĀ  H<sub>2</sub>O):


::: '''PreparationĀ  of 500 mM ADP stock solution with 300 mM freeĀ  Mg<sup>2+</sup>''' (dissolved in H<sub>2</sub>O):
::::# Take 1 g of ADP (can be orderd in portions of 1 g per vial).
Ā 
::::# Add 2.3 mL H<sub>2</sub>OĀ  (ADP is not dissolved at this stage).
:::: ToĀ  keep freeĀ  [Mg<sup>2+</sup>] constant during respirationĀ  measurementĀ  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 molĀ  MgCl<sub>2</sub>/mol ADP).
::::# Neutralize with 5 M KOH (approx. 900 Āµl). ADP will dissolve after addition of KOH.
Ā 
::::# Add 243.96 mg MgCl<sub>2</sub>. White precipitate will occur, which will dissolve during 1-2 min stirring or shaking.
:::: To find out more about the importance of adding Mg<sup>2+</sup>, please refer to [https://en.wikipedia.org/wiki/Magnesium_in_biology#Essential_role_in_the_biological_activity_of_ATP here]
::::# Check pH and adjust to 7 with 5 M KOH if necessary (usually about 30 to 50 Āµl). Addition of KOH causes precipitation. Carefully stir or shake it until the solution is clear again, then add the next droplet of KOH.
Ā 
::::# Adjust final volume to 4 mL and divide into 0.2 mL portions.
:::: '''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3)
::::# Store at -20 Ā°CĀ  Ā  Ā  Ā  Ā  Ā  (-80 Ā°C should be preferred for long-time storage).
Ā 
::::# Weigh 501.3 mg of ADP = 1 mmol ADP.
::::# Add 1.2 mL H<sub>2</sub>OĀ  (ADP is not dissolved at this stage).
::::# Neutralize with 5 M KOH (approx. 450 Āµl). ADP will dissolve after addition of KOH.
::::# Add 121.98 mg MgCl<sub>2</sub>. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT.
::::# Check pH and adjust to 7 with 5 M KOH if necessary.
::::# Adjust final volume to 2 mL and divide into 0.2 mL portions.
::::# Store at -80 Ā°C.
Ā 


:::: '''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 MgCl<sub>2</sub> solution (0.6 mol MgCl<sub>2</sub>/mol ADP) separately and perform a titration of both solutions in immediate succession.
:::: '''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 MgCl<sub>2</sub> solution (0.6 mol MgCl<sub>2</sub>/mol ADP) separately and perform a titration of both solutions in immediate succession.




::: '''PreparationĀ  of 200 mM ADP stock solution without Mg<sup>2+</sup> for experiments with [[Magnesium Green]]''' (dissolved inĀ  H<sub>2</sub>O):
:::: '''PreparationĀ  of 200 mM ADP stock solution without Mg<sup>2+</sup> for experiments with [[Magnesium Green]]''' (dissolved inĀ  H<sub>2</sub>O):


{{Template:200 mM ADP MgG}}
<!--{{Template:200 mM ADP MgG}}-->


::: '''O2k manual titrations'''Ā  [[MiPNet09.12 O2k-Titrations]]
:::Ā» '''O2k manual titrations'''Ā  [[MiPNet09.12 O2k-Titrations]]


:::: '''500 mM ADP:'''
:::: '''500 mM ADP:'''
::::* Titration volume: 4-20 ĀµL using a 25 ĀµL syringe (2 mL O2k-chamber).
::::* Titration volume ('''2-mL O2k-chamber'''): 4-20 ĀµL using a 25 ĀµL Hamilton syringe.
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1-5 ĀµL using a 10 ĀµL Hamilton syringe.
::::* Final concentration: 1-5 mM.
::::* Final concentration: 1-5 mM.


:::: '''200 mM ADP for experiments with [[Magnesium Green]]:'''
:::: '''200 mM ADP for experiments with [[Magnesium Green]]:'''
::::* Titration volume: 10-50 ĀµL using a 25 ĀµL syringe (2 mL O2k-chamber).
::::* Titration volume ('''2-mL O2k-chamber'''): 10-50 ĀµL using a 50 ĀµL Hamilton syringe.
::::* Final concentration: 1-5 mM.
::::* Final concentration: 1-5 mM.


== DatLab oxygen flux: performance and data analysis ==
== DatLab oxygen flux: performance and data analysis ==

Revision as of 07:35, 10 June 2021


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 (Calbiochem): 117105-1GM, store at 4 Ā°C, CAS: 72696-48-1, M = 501.3 gĀ·mol-1
Recommended due to lower ATP impurities, we use this (Merck-Calbiochem) ADP for preparation of 'ADP-Mg' solution in Oroboros Lab.
Alternative source: Sigma A 5285, 1 g, store at -20 Ā°C; M = 501.3 gĀ·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 gĀ·mol-1)
For instructions without MgCl2 and with smaller amounts of ADP see Talk:ADP


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 Āµl). 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 Āµl). 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 Ā°C (-80 Ā°C should be preferred for long-time storage).
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):


Ā» O2k manual titrations MiPNet09.12 O2k-Titrations
500 mM ADP:
  • Titration volume (2-mL O2k-chamber): 4-20 ĀµL using a 25 ĀµL Hamilton syringe.
  • Titration volume (0.5-mL O2k-chamber): 1-5 ĀµL using a 10 ĀµL Hamilton syringe.
  • Final concentration: 1-5 mM.
200 mM ADP for experiments with Magnesium Green:
  • Titration volume (2-mL O2k-chamber): 10-50 ĀµL using a 50 ĀµL Hamilton syringe.
  • 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 ā€˜State 3ā€™, ADP levels are ā€˜highā€™ (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.
Questions.jpg


Ā» Keywords
Specific
Ā» ANT
Ā» OXPHOS
Ā» OXPHOS capacity
Ā» Phosphorylation system
General
Ā» ATP
Ā» ATP synthase
Ā» MiR06
Ā» Mitochondrial preparations
Ā» O2k-Titrations


References

Bioblast linkReferenceYear
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 E et al ā€• MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v12020
Ost 2015 Fatty Acid Oxidation O2k-Network Discussion Forum
Selected media and chemicals for respirometry with mitochondrial preparations.
O2k-Protocols
2016-08-30
Vajda S, MƔndi M, KonrƠd 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
O2k-Protocols
O2k manual titrations: SUIT protocols with mitochondrial preparations.
2020-08-17


}}






MitoPedia topics: Substrate and metabolite