SUIT-014

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SUIT-014

Description

1GM;2D;3P;4S;5U;6Rot-.png

Abbreviation: NS(PGM)

Reference: A: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration Bioblast pdf »Versions

SUIT-category: NS(PGM)
SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot-

The SUIT-014 protocols are designed for cells or tissue types that display a preference for GM over PM to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the linear coupling control (L- P- E) with NADH linked-substrates (PGM) and the control in ET state (N, NS, S), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types.

Communicated by Cardoso LH, Doerrier C, Huete-Ortega M and Gnaiger E (last update 2019-02-26)

Specific SUIT protocols

1GM;2D;2c;3P;4S;5U;6Rot;7Ama.png D042 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1GM;2D;2c;3P;4S;5U;6Rot;7Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GM GML(n) N CI 1GM
2D GMP N CI 1GM;2D
2c GMcP N CI 1GM;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.
3P PGMP N CI 1GM;2D;2c;3P
4S PGMSP NS CI&II 1GM;2D;2c;3P;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
5U PGMSE NS CI&II 1GM;2D;2c;3P;4S;5U
6Rot SE S CII 1GM;2D;2c;3P;4S;5U;6Rot
7Ama ROX 1GM;2D;2c;3P;4S;5U;6Rot;7Ama
  • 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
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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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).
+ Mitochondrial external 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 c.
- It was observed that addition of P (5 mM) after GM inhibited respiration in horse skeletal muscle fibres (Votion et al 2012), which was not the case when P was titrated in steps of 1 mM.
- When evaluating the additive effect of the N- and S-pathway, it has to be considered that NSP- and NSE-capacities can only be compared with NP- and SE-capacities. This is not a problem when NSP = NSE (Gnaiger 2009). Otherwise, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.


Compare SUIT protocols

  • SUIT-011: 1GM;2D;2c;3S;4U;5Rot-; similar version of omitting P.
  • SUIT-012: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S.

References

 YearReferenceOrganismTissue;cell
Lemieux 2017 Sci Rep2017Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8.MouseHeart
Schoepf 2016 FEBS J2016Schöpf B, Schäfer G, Weber A, Talasz H, Eder IE, Klocker H, Gnaiger E (2016) Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells. FEBS J 283:2181-96.HumanEndothelial;epithelial;mesothelial cell
Genital
Other cell lines
Fibroblast

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry