SUIT-012

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-012

Description

1PM;2D;3G;4U-.png

Abbreviation: PM+G_OXPHOS

Reference: A: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM)

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

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


Communicated by Cardoso LHD, Doerrier C, Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-012 O2 mt D027

1PM;2D;2c;3G;4U;5Ama.png D027 O2 traces.png

  • SUIT-012 O2 mt D027 for mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)

SUIT-012 O2 ce-pce D052

Ce1;1Dig;1PM;2D;2c;3G;4U;5Ama.png D052 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

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


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
## 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). It is thus useful to understand the contribution and the activity of the dehydrogenases providing NADH.
+ 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

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry