SUIT-017

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SUIT-017

Description

1OctM;2D;2c;3G;4S;5U;6Rot-.png

Abbreviation: F+G+S_OXPHOS+Rot_ET

Reference: A:

SUIT protocol pattern: 1OctM;2D;3G;4S;5U;6Rot-

SUIT-017 gives information on F-pathway in LEAK state and OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated.

Communicated by Doerrier C, Huete-Ortega M, Iglesias-Gonzalez J, Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-017 O2 pfi D049

1OctM;2D;2c;3G;4S;5U;6Rot;7Ama.png D046 O2 traces.png

  • SUIT-017 O2 mt D046 evaluation of fatty acids and Q-junction additivity in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)

SUIT-017 O2 pfi D049

1OctM;2D;3G;4S;5U;6Rot;7Ama.png D049 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

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

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) FAO 1OctM

Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).

2D OctMP F(N) FAO 1OctM;2D

Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

2c OctMP F(N) FAO 1OctM;2D;2c

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]).Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

3G OctGMP FN F&CI 1OctM;2D;2c;3G

NADH-linked substrates (type N-pathway to Q). Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

4S OctGMSP FNS F&CI&II 1OctM;2D;2c;3G;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. Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

5U OctGMSE FNS F&CI&II 1OctM;2D;2c;3G;4S;5U

Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Noncoupled electron transfer state, ET-state, with ET-capacity, E.

6Rot SE S CII 1OctM;2D;2c;3G;4S;5U;6Rot

Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway if fatty acids are present. Noncoupled electron transfer state, ET-state, with ET-capacity, E.

7Ama ROX 1OctM;2D;2c;3G;4S;5U;6Rot;7Ama

Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.

Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


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Strengths and limitations

+ Glutamate is easier to prepare compared to pyruvate.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time by ~30 min.

Compare SUIT protocols

  • SUIT-002: A more comprehensive protocol including additional substrate states.
  • SUIT-005: A comparable protocol evaluating P instead of G.

References

 YearReferenceOrganismTissue;cell
Pesta 2012 Methods Mol Biol2012
O2k-Protocols
Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58.
HumanFibroblast
HEK
HUVEC
Other cell lines
Skeletal muscle

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry