Difference between revisions of "Pyruvate"
From Bioblast
| Line 26: | 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 == | == 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'' using Pyruvate, ADP, and Cytochrome C. However, I do not achieve a steady state level in OXPHOS. | :::: 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). | :::: 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 mor 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 2014 MitoPathways] | ||
:::: Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: [[O2k-Publications:_Drosophila]] | |||
::: | |||
: | |||
Revision as of 14:21, 20 April 2020
- high-resolution terminology - matching measurements at high-resolution
Pyruvate
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 need to combine pyruvate with at least a second NADH-linked substrate (e.g., Malate) or use a mor complex combination of substrates (e.g., Pyruvate&Glutamate&Malate). See Fig. 5.9. in Gnaiger 2014 MitoPathways
- Additionally, you may consult some of the publications from Drosophila melanogaster mitochondria: O2k-Publications:_Drosophila
