Pyruvate

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 pK_a = 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_m' 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 °C, CAS: 113-24-6, M = 110.0 g·mol^-1

Hazard statements: H317, H319; may cause an allergic skin reaction, causes serious eye irritation

It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 °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 µL, dissolved in H₂O) for use in 2-mL O2k-chamber:

1. Prepare fresh everyday.
2. Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
3. Add 180 µL H₂O.

» O2k manual titrations MiPNet09.12 O2k-Titrations

• Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
• Final concentration: 5 mM.

Preparation of 2.5 M stock solution (200 µL, dissolved in H₂O) for use in 0.5-mL O2k-chamber:

1. Prepare fresh everyday.
2. Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
3. Add 175 µL H₂O.

» O2k manual titrations MiPNet09.12 O2k-Titrations

• Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL 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