SUIT-012 O2 ce-pce D052

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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

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

The SUIT-012 O2 ce-pce D052 protocols specifically focus on assessing the linear coupling control (L- P- E) with NADH-linked substrates (PM) in permeabilized cells. Addition of G in NADH-supported OXPHOS enables evaluating the glutamate anaplerotic pathway control state. SUIT-012 O2 ce-pce D052 can be extended with the CIV assay module.

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

Steps and respiratory States

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
  • NADH-linked substrates (type N-pathway to Q).
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
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
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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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.

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References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry