SUIT-006

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SUIT-006

Description

1X;2D;2c;3Omy;4U;5Ama.png

Abbreviation: CCP-mtprep

Reference: A: Coupling control protocol, mtprep

SUIT protocol pattern: 1X;2D;3Omy;4U;5Ama

The SUIT-006 protocol is a coupling control protocol for mitochondrial preparations. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control (L- P- E or L- P-L(Omy)- E) at a defined electron transfer-pathway state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.

In the DatLab software, SUIT-006 DLP files are currently provided for the categories N(PM) and S. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created. For O2 application with ce-pce, choose SUIT-006_O2_ce-pce_D029 to create the DLPU, for mt, choose SUIT-006 O2 mt D047. For O2 and H2O2 measurements (AmR), choose SUIT-006 AmR mt D048, and for O2 and membrane potential measurements, choose SUIT-006 Fluo mt D034.


Communicated by Cardoso LH, Doerrier C, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-006 O2 ce-pce D029

Ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.png SUIT-006 O2 pce D029 traces.png

  • SUIT-006 O2 ce-pce D029 for N-pathway with permeabilized cells. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.

SUIT-006 O2 mt D047

1PM;2D;2c;3Omy;4U;5Ama.png SUIT-006 O2 mt D047.png

  • SUIT-006 O2 mt D047 for N-pathway with isolated mitochondria or tissue homogenate. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.

SUIT-006 AmR mt D048

1PM;2D;3Omy;4U;5Ama.png SUIT-006 AmR mt D048 O2.png SUIT-006 AmR mt D048.png

  • SUIT-006 AmR mt D048 to study simultaneously the O2 flux and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.

SUIT-006 Fluo mt D034

1PM;2D;3Omy;4U;5Ama.png SUIT-006 Fluo mt D034 O2.png SUIT-006 Fluo mt D034.png

  • SUIT-006 Fluo mt D034 to study simultaneously the O2 flux and mitochondrial membrane potential on isolated mitochondria, tissue homogenate and permeabilized cells.

SUIT-006 MgG mt D055

1PM;2D;3Cat;4U;5Ama.png SUIT-006 MgG mt D055 O2.png SUIT-006 MgG mt D055.png

  • SUIT-006 MgG mt D055 to study simultaneously the O2 flux and mitochondrial ATP production on isolated mitochondria, tissue homogenate and cells permeabilized previouslyto addition to the O2k chamber.

SUIT-006 O2 mt D022

1SRot;2D;2c;3(Omy);4U;5Ama.png SUIT-006 O2 mt D022 traces.png

  • SUIT-006 O2 mt D022 for S-pathway with isolated mitochondria, tissue homogenate and permeabilized cells.


MitoPedia: SUIT

Steps and respiratory states

1X;2D;2c;3Omy;4U;5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1X XL(n) 1X
  • X: combination of substrates and inhibitors chosen according to the aims.
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
2D XP 1X;2D
2c XcP 1X;2D;2c
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
  • 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]).
3Omy XLOmy 1X;2D;2c;3Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U XE 1X;2D;2c;3Omy;4U
5Ama ROX 1X;2D;2c;3Omy;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).


Questions.jpg


Click to expand or collaps

Strengths and limitations

+ Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
+ The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling control protocols for one specific ET-pathway state, e.g.: PM in the presence of Mna for N-pathway control state, succinate in the presence of rotenone for S-pathway control state. In the specific case of S-pathway control state, rotenone should be added to avoid reverse electron transfer (RET).
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- This protocol does not include internal ET-pathway control steps.
- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if applied (e.g., rotenone&succinate, SRot-pathway control state).
- LEAK respiration in the absence of Omy may be overestimated due to ATP turnover stimulated by endogenous adenylates. This can be evaluated by SUIT 1X;2D;3Omy;4U;-. In mouse brain homogenate with succinate&rotenone, L(n) and L(Omy) are identical (Krumschnabel et al 2014).
- CIV activity and Cytochrome c test cannot be performed together with the fluorescence (SUIT-006 AmR mt D048, SUIT-006 AmR mt D034 and SUIT-006 MgG mt D055).

Compare SUIT protocols

  • SUIT-001: Extended protocol (Reference protocol 1) with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
  • SUIT-004: Shortened version of SUIT-001, also with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
  • SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

References

 YearReferenceOrganismTissue;cell
Krumschnabel 2014 Methods Enzymol2014
O2k-Protocols contents
Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.
MouseNervous system
Votion 2012 PLoS One2012Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7:e34890.HorseSkeletal muscle


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry, Fluorometry