Difference between revisions of "Pyruvate"
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|mitopedia topic=Substrate and metabolite | |mitopedia topic=Substrate and metabolite | ||
}} | }} | ||
__TOC__ | |||
== Application in [[HRR]] == | == Application in [[HRR]] == | ||
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::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber). | ::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber). | ||
::::* Final concentration: 5 mM. | ::::* 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 will need to combine at least a 2<sup>nd</sup> NADH-linked substrate (''e.g.,'' Glutamate, Malate...). </div> | |||
* Additionally, you may consult some of the publications from ''Drosophila melanogaster'' mitochondria: | |||
https://wiki.oroboros.at/index.php/O2k-Publications:_Drosophila | |||
=== How to set a mark to calibrate the fluorescence signal using Amplex UltraRed assay? === | |||
::: 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 15:23, 5 March 2020
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 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)
- Prepare fresh everyday.
- Weigh 44 mg of pyruvic acid directyl into a 0.5 mL Eppendorf tube.
- Add 0.2 mL H2O.
- 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
- 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 will need to combine at least a 2nd NADH-linked substrate (e.g., Glutamate, Malate...).
- Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria:
https://wiki.oroboros.at/index.php/O2k-Publications:_Drosophila
How to set a mark to calibrate the fluorescence signal using Amplex UltraRed assay?
- To calibrate the fluorescence signal into µM, set the marks to the black line.
- -See:
- To calibrate the fluorescence signal into µM, set the marks to the black line.