Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Difference between revisions of "Pyruvate"

From Bioblast
Line 8: Line 8:
|mitopedia topic=Substrate and metabolite
|mitopedia topic=Substrate and metabolite
}}
}}
__TOC__
== Application in [[HRR]] ==
== Application in [[HRR]] ==


Line 24: Line 26:
::::* 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 14:23, 5 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 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...).
  • 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