ADP: Difference between revisions
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|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 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]]. | ||
|info=[[MiPNet03.02]], [[MiPNet09.12]] | |info=[[MiPNet03.02]], [[MiPNet09.12]] | ||
}}{{MitoPedia topics | |||
}} | |||
{{MitoPedia topics | |||
|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
}} | }} | ||
== 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, we use 'ADP-Mg' in [[AT_Innsbruck_OROBOROS |OROBOROS Lab]]. | Merck 117105-1GM ('''Calbiochem''') FW = 501.3; store at 4 °C. Recommended due to lower ATP impurities, we use 'ADP-Mg' in [[AT_Innsbruck_OROBOROS |OROBOROS Lab]]. | ||
Alternative source: Sigma A 5285, 1 g, store at -20 °C; FW = 501.3. | :::: Alternative source: Sigma A 5285, 1 g, store at -20 °C; FW = 501.3. | ||
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> | :::: <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): | ::: '''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 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): | ::: '''Preparation of 500 mM ADP stock solution with 300 mM free Mg<sup>2+</sup>''' (dissolved in H<sub>2</sub>O): | ||
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 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). | ||
'''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3) | :::: '''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3) | ||
:: | ::::# 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. | ||
:: | ::::# 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 if necessary. | ||
:: | ::::# Adjust final volume to 2 ml and divide into 0.2 ml portions. | ||
:: | ::::# Store at -80 °C. | ||
::'''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. | :::: '''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. | ||
'''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ::: '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ||
::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber). | ::::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber). | ||
::* Final concentration: 1-5 mM. | ::::* Final concentration: 1-5 mM. |
Revision as of 18:02, 6 November 2016
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.
Abbreviation: D
Reference: MiPNet03.02, MiPNet09.12
MitoPedia topics: Substrate and metabolite
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):
- Weigh 501.3 mg of ADP = 1 mmol ADP
- Add 1.2 ml H2O (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 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 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).
- MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)
- Weigh 501.3 mg of ADP = 1 mmol ADP.
- Add 1.2 ml H2O (ADP is not dissolved at this stage).
- Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
- Add 121,98 mg MgCl2. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT.
- Check pH and adjust to 7 if necessary.
- Adjust final volume to 2 ml and divide into 0.2 ml portions.
- Store at -80 °C.
- 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 MgCl2 solution (0.6 mol MgCl2/mol ADP) seperately and perform a titration of both solutions in immediate succession.
- O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
- Final concentration: 1-5 mM.