- high-resolution terminology - matching measurements at high-resolution
Talk:SUIT-029
Description
Abbreviation: PM_OXPHOS+Rot_ET
Reference: A: quality control evaluation of mitochondrial preparations - SUIT-029
O2k-Application: O2
The SUIT-029 protocols are designed to to evaluate the quality of mitochondrial preparations taking into account the presence of ATPases with N substrates) (PM) without the additive effect of the S-pathway, which would decrease the coupling degree. Additionally, PM provides an higher N-pathway contribution when compared to the use of GM substrates. In this way, it comprises 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.
SUIT-029 protocols also include the assessment of mitochondrial outer membrane integrity ( 2c) and the LEAK respiration overestimation is prevented in the presence of Oligomycin, without the uncoupling promoted by the ATPase contamination and as a control with the L(n) previously obtained.
- + Internal ET-pathway control step is included as a test for limitation of OXPHOS-capacity by the phosphorylation system relative to ET-capacity. The evaluation of ET-state with NADH-linked substrates allows to calculate ET-coupling efficiency (1-L/E).
- + The additive effect of convergent flux through NADH-linked respiration in ET-state is evaluated (5G).
- + ET-state with NS-linked substrates is evaluated (5S) followed by additional U titration to ensure ET-capacity.
- + Assessment of ET-capacity not only in N- and NS-pathway but also in S-pathway (7Rot).
- + This protocol can be optionally harmonized with many other SUIT protocols (e.g., SUIT-001, SUIT-004, SUIT-008). Addition of G in NADH-supported OXPHOS enables evaluation of the glutamate anaplerotic pathway control state.
- + This protocol can be extended with the Complex IV module.
- - Long duration of the experiment due to adjustment of Oligomycin concentration (in the case of a new isolation protocol). For a shorter protocol the titration of glutamate, succinate and rotenone could be omitted.
- - The use of Oligomycin could affect the evaluation of the ET capacity (could inhibit ET-state).
- - F-pathway is not analyzed.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
