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Difference between revisions of "ADP"

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(moved old instructions to the Talk-page, updated instructions to what we always do: take 1 g ADP that comes in a small vial instead of weighting it. Also moved comments about Mg to paragraph above)
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{{MitoPedia
{{MitoPedia
|abbr=D
|abbr=D
|description='''Adenosine diphosphate''' is a nucleotid. 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]]
|type=Substrate PS
__TOC__
}}
 
{{MitoPedia concepts|type=Substrate PS
}}
{{MitoPedia methods|type=Substrate PS
}}
{{MitoPedia O2k and high-resolution respirometry|type=Substrate PS
}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
|type=Substrate PS
}}
== Application in [[HRR]] ==
== Application in [[HRR]] ==


'''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),  
::: '''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),  
Merck  117105-1GM ('''Calbiochem''') FW = 501.3; store at 4 °C. Recommended due to lower ATP impurities and used in [[AT_Innsbruck_OROBOROS |OROBOROS Lab]].
::: Merck  117105-1GM ('''Calbiochem''') M = 501.3 g·mol<sup>-1</sup>; store at 4 °C. Recommended due to lower ATP impurities, we use this ADP for preparation of 'ADP-Mg' solution  in [[AT_Innsbruck_Oroboros |Oroboros Lab]].
::: Alternative source:  Sigma A 5285, 1 g, store at -20 °C; M = 501.3 g·mol<sup>-1</sup>.
 
:::: To  keep free  [Mg<sup>2+</sup>] constant during respiration  measurement  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 mol  MgCl<sub>2</sub>/mol ADP).
:::: 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 g·mol<sup>-1</sup>)
:::: For instructions without MgCl<sub>2</sub> and with smaller amounts of ADP see [[Talk:ADP]]
 
:::: <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
 
<!--
:::: '''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O):
 
::::# Weigh 501.3 mg of ADP = 1 mmol ADP
::::# Add 1.2 mL H<sub>2</sub>O  (ADP is not dissolved at this stage).
::::# Neutralize with 5 M KOH (approx. 450 µL). ADP will dissolve after addition of KOH.
::::# 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 °C.
-->
 
:::: '''Preparation  of 500 mM ADP stock solution with 300 mM free  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).
::::# Add 2.3 mL H<sub>2</sub>O  (ADP is not dissolved at this stage).
::::# Neutralize with 5 M KOH (approx. 900 µl). ADP will dissolve after addition of KOH.
::::# Add 243.96 mg MgCl<sub>2</sub>. White precipitate will occur, which will dissolve during 1-2 min stirring or shaking.
::::# 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.
::::# Adjust final volume to 4 mL and divide into 0.2 mL portions.
::::# 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 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.




Alternative sourceSigma A 5285, 1 g, store at -20 °C; FW = 501.3.
:::: '''Preparation of 200 mM ADP stock solution without Mg<sup>2+</sup> for experiments with [[Magnesium Green]]''' (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>
<!--{{Template:200 mM ADP MgG}}-->


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


'''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O):
:::: '''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.


::1) Weigh 501.3 mg of ADP = 1 mmol ADP
:::: '''200 mM ADP for experiments with [[Magnesium Green]]:'''
::2) Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
::::* Titration volume ('''2-mL O2k-chamber'''): 10-50 µL using a 50 µL Hamilton syringe.
::3) Neutralize with 5 N KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
::::* Final concentration: 1-5 mM.
::4) Check pH and adjust to 7 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  Mg<sup>2+</sup>''' (dissolved in  H<sub>2</sub>O):
== 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]]


To  keep free  [Mg<sup>2+</sup>] constant during respiration measurement  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 mol  MgCl<sub>2</sub>/mol ADP).
== ADP dependence of respiration ==


'''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3)
:::: 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_1955_JBC-III|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_2000_Proc_Natl_Acad_Sci_U S A|Gnaiger et al 2000]] and [[Gnaiger_2001_Respir_Physiol|Gnaiger 2001]].


::1) Weigh 501.3 mg of ADP = 1 mmol ADP.
[[File:Questions.jpg|left|40px]]
::2) Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
<br />
::3) Neutralize with 5 N KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
<div class="toccolours mw-collapsible mw-collapsed">
::4)  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.
::: <span style="font-size:105%; color:##424242">'''» Keywords'''</span>
::5) Check pH and adjust to 7 if necessary.
<div class="mw-collapsible-content">
::6) Adjust final volume to 2 ml and divide into 0.2 ml portions.
::: '''Specific'''
::7) Store at -80 °C.
::::» [[ANT]]
::::» [[OXPHOS]]
::::» [[OXPHOS capacity]]
::::» [[Phosphorylation system]]


::: '''General'''
::::» [[ATP]]
::::» [[ATP synthase]]
::::» [[MiR06]]
::::» [[Mitochondrial preparations]]
::::» [[MiPNet09.12 O2k-Titrations|O2k-Titrations]]
</div>
</div>
<br />


::'''Comment''': In some cases it was observed, that after defreezing of the 'ADP-Mg' a white precipitate has formed again. 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) seperately and perform a titration of both solutions in immediate succession.
== References ==
{{#ask:[[Additional label::ADP]]
| mainlabel=Bioblast link
|?Has title=Reference
|?Was published in year=Year
|format=broadtable
|limit=5000
|offset=0
|sort=Has title
|order=ascending
}}




'''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
}}
{{MitoPedia concepts}}
{{MitoPedia methods}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
}}


::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
[[Category:Coupling control]]
::* Final concentration: 1-5 mM.

Revision as of 11:58, 24 February 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 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

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.
Questions.jpg



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 ― 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, 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
O2k-Protocols
O2k manual titrations: SUIT protocols with mitochondrial preparations.
2020-08-17






MitoPedia topics: Substrate and metabolite