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Difference between revisions of "Pyruvate"

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(replaced FW by M and added units g*mol-1)
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|description=[[File:Pyruvic_acid.jpg|left|80px|Pyruvic acid]]
|description=[[File:Pyruvic_acid.jpg|left|80px|Pyruvic acid]]
'''Pyruvic acid''', C<sub>3</sub>H<sub>4</sub>O<sub>3</sub>, is an alpha-keto monocarboxylic acid which occurs under physiological conditions mainly as the anion '''pyruvate<sup>-</sup>, P''', with ''p''K<sub>a</sub> = 2.5. Pyruvate is formed in glycolysis from phosphoenolpyruvate. In the cytosol, pyruvate is a substrate of [[lactate dehydrogenase]]. Pyruvate enters the mitochondrial matrix via a specific low ''K''<sub>m</sub>' H<sup>+</sup>/monocarboxylate cotransporter known as the [[pyruvate carrier]]. Similarly, the plasma membrane of many cell types has H<sup>+</sup>/monocarboxylate cotransporter activity and pyruvate can thus be added as a substrate to living cells. In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by [[pyruvate dehydrogenase]] and yields [[acetyl-CoA]]. Pyruvate competitively reverses the inhibition of [[Complex IV | cytochrome ''c'' oxidase]] by [[cyanide]]. Pyruvate is an antioxidant reacting with [[hydrogen peroxide]].
'''Pyruvic acid''', C<sub>3</sub>H<sub>4</sub>O<sub>3</sub>, is an alpha-keto monocarboxylic acid which occurs under physiological conditions mainly as the anion '''pyruvate<sup>-</sup>, P''', with ''p''K<sub>a</sub> = 2.5. Pyruvate is formed in glycolysis from phosphoenolpyruvate. In the cytosol, pyruvate is a substrate of [[lactate dehydrogenase]]. Pyruvate enters the mitochondrial matrix via a specific low ''K''<sub>m</sub>' H<sup>+</sup>/monocarboxylate cotransporter known as the [[pyruvate carrier]]. Similarly, the plasma membrane of many cell types has H<sup>+</sup>/monocarboxylate cotransporter activity and pyruvate can thus be added as a substrate to living cells. In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by [[pyruvate dehydrogenase]] and yields [[acetyl-CoA]]. Pyruvate competitively reverses the inhibition of [[Complex IV | cytochrome ''c'' oxidase]] by [[cyanide]]. Pyruvate is an antioxidant reacting with [[hydrogen peroxide]].
|info=[[Gnaiger 2014 MitoPathways]], [[MiPNet09.12 O2k-Titrations]]
|info=[[Gnaiger 2020 BEC MitoPathways]], [[MiPNet09.12 O2k-Titrations]]
}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
}}
}}
__TOC__
__TOC__
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== Application in [[HRR]] ==
== Application in [[HRR]] ==


::: '''P: Pyruvate''' (pyruvic acid, sodium salt, C<sub>3</sub>H<sub>3</sub>O<sub>3</sub>Na); Sigma P 2256, 25 g, store at 4 °C; FW = 110.0
::: '''P: Pyruvate''' (pyruvic acid, sodium salt, C<sub>3</sub>H<sub>3</sub>O<sub>3</sub>Na); Sigma P 2256, 25 g, store at 4 °C; M = 110.0 g·mol<sup>-1</sup>
:::: It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 °C, to be diluted only on the day of use.


::: '''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O)
 
:::: '''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O) for use in '''2-mL O2k-chamber''':


::::# Prepare fresh everyday.
::::# Prepare fresh everyday.
::::# Weigh 44 mg of pyruvic acid directyl into a 0.5 mL Eppendorf tube.
::::# Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
::::# Add 0.2 mL H<sub>2</sub>O.
::::# Add 0.2 mL H<sub>2</sub>O.
::::# Adjust pH to 7.0.
::::# Adjust pH to 7.0.


:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
::::* Final concentration: 5 mM.
:::: '''Preparation of 2.5 M stock solution''' (dissolved in H<sub>2</sub>O)  for use in '''0.5-mL O2k-chamber''':
::::# Prepare fresh everyday.
::::# Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
::::# Add 0.2 mL H<sub>2</sub>O.
::::# Adjust pH to 7.0.


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


::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 5 mM.
::::* Final concentration: 5 mM.
== Troubleshooting ==
== Troubleshooting ==
=== Unstable respiration while using Pyruvate as the only substrate ===
=== Unstable respiration while using pyruvate as the only substrate ===
* '''Customer ID''': [https://www.bioblast.at/index.php/AU_Melbourne_White_C AU Melbourne White C]
:::* '''Customer ID''': [https://www.bioblast.at/index.php/AU_Melbourne_White_C AU Melbourne White C]
'''Question:'''
::::'''Question:'''


:::: I am evaluating mitochondrial respiration from ''Drosophila melanogaster'' using  Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS.
:::: I am evaluating mitochondrial respiration from ''Drosophila melanogaster'' using  Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS.


Any advice would be appreciated. The data is attached (2019-07-17).
:::: Any advice would be appreciated. The data is attached (2019-07-17).


[[File:Ticket2019072631000015.png|700px|center]]
[[File:Ticket2019072631000015.png|700px|center]]


* <div>'''Answer:'''</div><div>Pyruvate alone is not sufficient to support NADH-linked respiration. In order to do so you will need to combine at least a 2<sup>nd</sup> NADH-linked substrate (''e.g.,'' Glutamate, Malate...). </div>
:::* <div>'''Answer:'''</div><div>Pyruvate alone is not sufficient to support NADH-linked respiration. In order to do so you need to combine pyruvate with at least a second NADH-linked substrate (e.g. Malate) or use a more complex combination of substrates (''e.g.,'' Pyruvate&Glutamate&Malate). See Fig. 5.9. in [https://wiki.oroboros.at/images/f/fc/Gnaiger_2014_Mitochondr_Physiol_Network_MitoPathways.pdf Gnaiger 2020 BEC MitoPathways]
* Additionally, you may consult some of the publications from ''Drosophila melanogaster'' mitochondria:


https://wiki.oroboros.at/index.php/O2k-Publications:_Drosophila
:::: Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: [[O2k-Publications:_Drosophila]]


 
{{MitoPedia topics
=== How to set a mark to calibrate the fluorescence signal using Amplex UltraRed assay? ===
|mitopedia topic=Substrate and metabolite
::: To calibrate the fluorescence signal into µM, set the marks to the black line.
}}
:::*''-See:''
::::[[MiPNet20.14 AmplexRed H2O2-production]]
::::[[Amp calibration - DatLab]]

Revision as of 12:02, 12 February 2021


high-resolution terminology - matching measurements at high-resolution


Pyruvate

Description

Pyruvic acid

Pyruvic acid, C3H4O3, is an alpha-keto monocarboxylic acid which occurs under physiological conditions mainly as the anion pyruvate-, P, with pKa = 2.5. Pyruvate is formed in glycolysis from phosphoenolpyruvate. In the cytosol, pyruvate is a substrate of lactate dehydrogenase. Pyruvate enters the mitochondrial matrix via a specific low Km' H+/monocarboxylate cotransporter known as the pyruvate carrier. Similarly, the plasma membrane of many cell types has H+/monocarboxylate cotransporter activity and pyruvate can thus be added as a substrate to living cells. In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase and yields acetyl-CoA. Pyruvate competitively reverses the inhibition of cytochrome c oxidase by cyanide. Pyruvate is an antioxidant reacting with hydrogen peroxide.

Abbreviation: P

Reference: Gnaiger 2020 BEC MitoPathways, MiPNet09.12 O2k-Titrations

Application in HRR

P: Pyruvate (pyruvic acid, sodium salt, C3H3O3Na); Sigma P 2256, 25 g, store at 4 °C; M = 110.0 g·mol-1
It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 °C, to be diluted only on the day of use.


Preparation of 2 M stock solution (dissolved in H2O) for use in 2-mL O2k-chamber:
  1. Prepare fresh everyday.
  2. Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
  3. Add 0.2 mL H2O.
  4. Adjust pH to 7.0.
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
  • Final concentration: 5 mM.


Preparation of 2.5 M stock solution (dissolved in H2O) for use in 0.5-mL O2k-chamber:
  1. Prepare fresh everyday.
  2. Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
  3. Add 0.2 mL H2O.
  4. Adjust pH to 7.0.
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 5 mM.


Troubleshooting

Unstable respiration while using pyruvate as the only substrate

Question:
I am evaluating mitochondrial respiration from Drosophila melanogaster using Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS.
Any advice would be appreciated. The data is attached (2019-07-17).
Ticket2019072631000015.png
  • Answer:
    Pyruvate alone is not sufficient to support NADH-linked respiration. In order to do so you need to combine pyruvate with at least a second NADH-linked substrate (e.g. Malate) or use a more complex combination of substrates (e.g., Pyruvate&Glutamate&Malate). See Fig. 5.9. in Gnaiger 2020 BEC MitoPathways
Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: O2k-Publications:_Drosophila


MitoPedia topics: Substrate and metabolite