SUIT-008 O2 mt D026

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SUIT-008 O2 mt D026

Description

1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png

Abbreviation: Q-junction mtprep

Reference: A: SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - SUIT-008

SUIT number: D026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern: 1PM;2D;3G;4S;5U;6Rot-

The SUIT-008 O2 mt D026 protocol can be used with mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of organisms and tissues. The protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 mt D026 can be easily extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-06-06)
MitoPedia: SUIT

Steps and respiratory states

SUIT-008

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
4S PGMSP NS CI&II 1PM;2D;2c;3G;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
5U PGMSE NS CI&II 1PM;2D;2c;3G;4S;5U
6Rot SE S CII 1PM;2D;2c;3G;4S;5U;6Rot
7Ama ROX 1PM;2D;2c;3G;4S;5U;6Rot;7Ama
  • 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


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Strengths and limitations

+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
+ The presence of PGM and S establishes fully operative TCA cycle activity.
+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
+ Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome c (cytochrome c test). The early addition of cytochrome c in the protocol ensures comparability of all states in case of any effect of cytochrome c.
+ 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 S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for the diagnosis of N-capacity.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- F-pathway is not analysed.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-004: SUIT-004 protocol provide a quick assessment of the linear coupling control (L-P-E) with NADH linked-substrates (PM) and the pathway control in ET state (N, NS, S pathways).
  • SUIT-011: SUIT-011 protocol are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS capacity with GMS as NS-linked substrates).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry