SUIT-012 O2 ce-pce D052

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MitoPedia

SUIT-012 O2 ce-pce D052

Description

Ce1;1Dig;1PM;2D;2c;3G;4U;5Ama.png

Abbreviation: N(PGM)

Reference: A Linear coupling control with NADH-linked substrates for pce - SUIT-012

SUIT number: D052_ce1;1Dig;1PM;2D;2c;3G;4U;5Ama

O2k-Application: O2

SUIT-category: N(PGM)
SUIT protocol pattern: diametral ce1;1Dig;1PM;2D;2c;3G;4U-

The SUIT-012 O2 ce-pce D052 protocol specifically focuses on assessing the coupling control (L- P- E) with NADH-linked substrates (PM and PGM). Addition of G enables evaluating the contribution of this substrate in NADH-supported OXPHOS respiration, which may be relevant in the presence of glutamate-anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.

Communicated by Huete-Ortega M, Komlodi T, Gnaiger E (last update 2019-04-03)

Steps and respiratory States

Representative traces

D052 O2 traces.png

MitoPedia: SUIT

Ce1;1Dig;1PM;2D;2c;3G;4U;5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig ROX ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
3G PGMP N CI 1PM;2D;2c;3G
4U PGME N CI 1PM;2D;2c;3G;4U
5Ama ROX 1PM;2D;2c;3G;4U;5Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


Click to expand or collaps

Strengths and limitations

+ This protocol allows to evaluate the function of the TCA cycle without the involvement of the complex II (S-pathway). Would be useful to understand the contribution and the activity of the dehydrogenases.
+ Mitochondrial outer membrane integrity can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of cytochrome c.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and of the S-pathway is higher with GM compared to PM (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for diagnosis of N-capacity.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-008: 1PM;2D;3G;4S;5U-; longer version that also analyses S-pathway.
  • SUIT-006: 1X;2D;3Omy;4U-; coupling control protocol with only one combination of substrates (e.g. 1PM;2D;3Omy;4U-).

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3G Glutamate (G)
4U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry