SUIT-012 O2 mt D027

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         MitoPedia: SUIT         MiP and biochemistry         Preprints and history


SUIT-012 O2 mt D027



Abbreviation: N CCP mtprep

Reference: A Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-012

SUIT number: D027_1PM;2D;2c;3G;4U;5Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern: 1PM;2D;2c;3G;4U-

The SUIT-012 O2 mt D027 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, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-06)

Representative traces

D027 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
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
## Azd ROX


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). It can 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-).


MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry