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
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 species 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)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
- » Selected keywords in SUIT protocols
- Pathway control
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).
MitoPedia methods: Respirometry