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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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|abbr=P
|abbr=P
|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 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>' transporter known as the [[pyruvate carrier]].
'''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]].
In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by [[pyruvate dehydrogenase]] and yields [[acetyl-CoA]].
|info=[[Gnaiger 2020 BEC MitoPathways]], [[MiPNet09.12 O2k-Titrations]]
Pyruvate competitively reverses the inhibition of [[Complex IV | cytochrome ''c'' oxidase]] by cyanide. Pyruvate is an antioxidant reacting with [[hydrogen peroxide]].
|info=[http://www.oroboros.at/?Gnaiger_2012_MitoPathways Gnaiger 2012 MitoPathways], [[MiPNet09.12]]
|type=Substrate ETS
}}
{{MitoPedia methods|type=Substrate ETS
}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
|type=Substrate ETS
}}
}}
__TOC__
== 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)  for use in '''2-mL O2k-chamber''':


::::# Prepare fresh everyday.
::::# Weigh 44 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.


'''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O)
:::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]]
: Prepare fresh everyday.


::1) Weigh 44 mg of pyruvic acid directyl into a 1 ml Eppendorf tube.
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
::2) Add 0.2 ml H<sub>2</sub>O.
::::* Final concentration: 5 mM.
::3) Adjust pH to 7.0.




'''Oxygraph-2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
:::: '''Preparation of 2.5 M stock solution''' (dissolved in H<sub>2</sub>O) for use in '''0.5-mL O2k-chamber''':


::* Titration volume: 5 µl using a 25 µl syringe (2 ml O2k-chamber).
::::# Prepare fresh everyday.
::* Final concentration: 5 mM.
::::# 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]]
 
::::* 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 ===
:::* '''Customer ID''': [https://www.bioblast.at/index.php/AU_Melbourne_White_C AU Melbourne White C]
::::'''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).
 
[[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 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: [[O2k-Publications:_Drosophila]]
 
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
}}

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