Pyruvate: Difference between revisions
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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 | |info=[[Gnaiger 2020 BEC MitoPathways]], [[MiPNet09.12 O2k-Titrations]] | ||
}} | }} | ||
__TOC__ | __TOC__ | ||
== Application in [[HRR]] == | == Application in [[HRR]] == | ||
{{Chemical_description | |||
|abbr=P | |||
|trivial name=Pyruvate | |||
|complete name=pyruvic acid, sodium salt | |||
|chem formula=C<sub>3</sub>H<sub>3</sub>O<sub>3</sub>Na | |||
|molar mass=110.0 | |||
|vendor=Sigma-Aldrich | |||
|product number=P2256 | |||
|store at=4 Β°C | |||
|sensitivity= | |||
|cas=113-24-6 | |||
|h statements=H317, H319 | |||
|h info=may cause an allergic skin reaction, causes serious eye irritation | |||
}}<!--::: '''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 Β°C; M = 110.0 gΒ·mol<sup>-1</sup>--> | |||
:::: It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 Β°C, to be diluted only on the day of use. | |||
:::: After addition of H<sub>2</sub>O the pH of the Pyruvate solution is about 6. This is acceptable without pH-adjustment, because the titrated volumes are small and reaction media are buffered. | |||
:::: 2021-03: The preparation instructions were updated to take the volume of the solute (P) into account (see: [[Volume_of_the_solute| Volume of the solute]]). The concentrations prepared following the former instructions (see Discussion section) are sufficiently high for SUIT protocol titrations. | |||
::: '''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O) | :::: '''Preparation of 2 M stock solution''' (200 Β΅L, 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 | ::::# Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. | ||
::::# Add | ::::# Add 180 Β΅L H<sub>2</sub>O. | ||
:::: | Β | ||
:::Β» '''O2k manual titrations'''Β [[MiPNet09.12 O2k-Titrations]] | |||
Β | |||
::::* Titration volume ('''2-mL O2k-chamber'''): 5 Β΅L using a 25 Β΅L Hamilton syringe. | |||
::::* Final concentration: 5 mM. | |||
::: ''' | :::: '''Preparation of 2.5 M stock solution''' (200 Β΅L, dissolved in H<sub>2</sub>O) for use in '''0.5-mL O2k-chamber''': | ||
::::* Titration volume: | ::::# Prepare fresh everyday. | ||
::::# Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. | |||
::::# Add 175 Β΅L H<sub>2</sub>O. | |||
:::Β» '''O2k manual titrations'''Β [[MiPNet09.12 O2k-Titrations]] | |||
Β | |||
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 Β΅L using a 10 Β΅L Hamilton syringe. | |||
::::* Final concentration: 5 mM. | ::::* Final concentration: 5 mM. | ||
== Troubleshooting == | == Troubleshooting == | ||
=== Unstable respiration while using | === 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'' usingΒ Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS. | :::: I am evaluating mitochondrial respiration from ''Drosophila melanogaster'' usingΒ 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 | :::* <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 | :::: Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: [[O2k-Publications:_Drosophila]] | ||
{{MitoPedia topics | |||
Β | |mitopedia topic=Substrate and metabolite | ||
Β | }} | ||
Latest revision as of 11:20, 17 June 2021
Description
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-Aldrich: P2256, store at 4 Β°C, CAS: 113-24-6, M = 110.0 gΒ·mol-1
- Hazard statements: H317, H319; may cause an allergic skin reaction, causes serious eye irritation
- P: Pyruvate (pyruvic acid, sodium salt; C3H3O3Na), Sigma-Aldrich: P2256, store at 4 Β°C, CAS: 113-24-6, M = 110.0 gΒ·mol-1
- It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 Β°C, to be diluted only on the day of use.
- After addition of H2O the pH of the Pyruvate solution is about 6. This is acceptable without pH-adjustment, because the titrated volumes are small and reaction media are buffered.
- 2021-03: The preparation instructions were updated to take the volume of the solute (P) into account (see: Volume of the solute). The concentrations prepared following the former instructions (see Discussion section) are sufficiently high for SUIT protocol titrations.
- Preparation of 2 M stock solution (200 Β΅L, dissolved in H2O) 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 180 Β΅L H2O.
- Β» O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 Β΅L using a 25 Β΅L Hamilton syringe.
- Final concentration: 5 mM.
- Preparation of 2.5 M stock solution (200 Β΅L, dissolved in H2O) 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 175 Β΅L H2O.
- Β» O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 1 Β΅L using a 10 Β΅L Hamilton syringe.
- Final concentration: 5 mM.
Troubleshooting
Unstable respiration while using pyruvate as the only substrate
- Customer ID: 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).
- 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