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Difference between revisions of "Pyruvate"

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{{MitoPedia
{{MitoPedia
|abbr=P
|abbr=P
|description=Pyruvate as a product of glycolysis is an anion that enters mitochondrial matrix via a specific low Km transporter [[pyruvate carrier]].
|description=[[File:Pyruvic_acid.jpg|left|80px|Pyruvic acid]]
In the mt matrix the oxidative decarboxylation of '''pyruvate''' is catalyzed by [[pyruvate dehydrogenase complex]] and yields acetyl-CoA.
'''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 alone cannot support respiration of [[Mitochondrial preparations|mt-preparations]] from most tissues. 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]]
|info=[[Gnaiger 2014 MitoPathways]], [[MiPNet09.12 O2k-Titrations]]
|type=Substrate ETS
}}
{{MitoPedia methods|type=Substrate ETS
}}
}}
{{MitoPedia topics
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
|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; FW = 110.0
 
::: '''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O)
 
::::# Prepare fresh everyday.
::::# Weigh 44 mg of pyruvic acid directyl into a 0.5 mL Eppendorf tube.
::::# Add 0.2 mL H<sub>2</sub>O.
::::# Adjust pH to 7.0.


<span style="color:#8B008B"> '''Caution:''' Prepare fresh everyday!</span>


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


'''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O):
::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
::::* 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:'''


::1) weigh 44 mg of L-Glutamic acid directyl into 1 ml Eppendorf tube
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.
::2) add 0.2 ml H<sub>2</sub>O
::3) adjust pH to 7


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


'''Oxygraph-2k Manual Titrations:'''  [[MiPNet09.12]]
[[File:Ticket2019072631000015.png|700px|center]]


::* Titration volume: 5 µl using a 25 µl syringe
'''Answer:'''
::* Final conc. in 2 ml O2k-chamber: 5 mM
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...). ''See'' [[Gnaiger 2014 MitoPathways]]
Additionally, you may consult some of the publications from ''Drosophila melanogaster'' mitochondria:[[O2k-Publications:_Drosophila]]

Revision as of 13:38, 20 March 2020


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 alone cannot support respiration of mt-preparations from most tissues. Pyruvate competitively reverses the inhibition of cytochrome c oxidase by cyanide. Pyruvate is an antioxidant reacting with hydrogen peroxide.

Abbreviation: P

Reference: Gnaiger 2014 MitoPathways, MiPNet09.12 O2k-Titrations


MitoPedia topics: Substrate and metabolite 

Application in HRR

P: Pyruvate (pyruvic acid, sodium salt, C3H3O3Na); Sigma P 2256, 25 g, store at 4 °C; FW = 110.0
Preparation of 2 M stock solution (dissolved in H2O)
  1. Prepare fresh everyday.
  2. Weigh 44 mg of pyruvic acid directyl 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: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
  • 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 will need to combine at least a 2nd NADH-linked substrate (e.g., Glutamate, Malate...). See Gnaiger 2014 MitoPathways

Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria:O2k-Publications:_Drosophila