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
The SUIT-012 O2 mt D027 protocols specifically focus on assessing the linear coupling control (L- P- E) with NADH-linked substrates (PM) in 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. Addition of G in NADH-supported OXPHOS enables evaluating the glutamate anaplerotic pathway control state. SUIT-012 O2 mt D027 can be extended with the CIV assay module.
Communicated by Huete-Ortega M, Iglesias-Gonzalez J 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
- + 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.
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