SUIT protocol library

Substrate-uncoupler-inhibitor titration (SUIT) protocols

Classes of SUIT protocols with coupling and substrate control defined for mitochondrial preparations

This page is in preparation (intern wiki: [1])

Strategy

1. Select a pathway control state.
2. Select a control pattern.
3. Define substrates for each pathway state.
4. Define coupling states.

Definition of states

Experimental conditions

• Temp
• O2
• Medium
• Oxygen level
• ADP kinetics
• Substrate kinetics

O2k configuration

• Amp: Amplex Red, TMRM, Ca Green, ...
• Pot
• Spectrophotometry

Define substrates

Substrate control states are defined for each pathway control state (CI-linked, CII-linked, CI&II-linked) is defined by a specific substrate or substrate combination. These substrates may be added in a single step, or in multiple steps for analyis of the effect of different substrate combinations within a pathway control state.

• CI: PM, GM, PGM, M, ..
• CII: S(Rot), S, SM, ..
• CI&II: PS, GS, PMS, GMS, PGMS, ..
• CIV: AsTM
• FAO: Oct, Pal, ...
• CGPDH:
• c

Define coupling states

The coupling control states require a complementary definition of an ETS-competent substrate state.

• L, P, E (Uncouplers, U: FCCP, CCCP, DNP)

Coupling states:

Define residual oxygen consumption

• ROX: Rot&Ama, Rot&Mna&Ama, Myx, Cn, Az, ..

Pathway control states

Single pathway control states

1. CI
2. CII
3. CI&CII
4. CIV
5. FAO
6. CGPDH

Multiple pathway control states

A. CI-CII-CIV

1. CI + CII
2. CI + CII + CIV
3. CI + CI&CII + CII
4. CI + CI&CII + CII + CIV

B. FAO-CI-CII-CIV

1. FAO + CI&FAO + CII
2. FAO + CI&FAO + CII + CIV
3. FAO + CI&FAO + CI&CII&FAO + CII
4. FAO + CI&FAO + CI&CII&FAO + CII + CIV

C. CGPDH-CI-CII-CIV

1. CGPDH + CI&CGPDH
2. CGPDH + CI&CGPDH + CII&CGPDH
3. CGPDH + CI&CGPDH + CII&CGPDH + CIV
4. CGPDH + CI&CGPDH + CI&CII&CGPDH + CII&CGPDH
5. CGPDH + CI&CGPDH + CI&CII&CGPDH + CII&CGPDH + CIV
6. CI + CI&CGPDH + ...
7. CI + CI&CII + CI&CII&CGPDH + ...
8. CI&CII + CI&CII&CGPDH + ...

D. FAO-CGPDH-CI-CII-CIV

1. FAO + FAO&CGPDH + CI&FAO&CGPDH + ...
2. CGPDH + FAO&CGPDH + CI&FAO&CGPDH + ...
3. CI + CI&FAO + CI&FAO&CGPDH + ...
4. CI + CI&CGPDH + CI&FAO&CGPDH + ...
5. ...

Control patterns

Linear

1. Linear coupling control: at a single substrate state (classical State 3 -> State 4, or P -> L_T).
2. Linear substrate control: at a single coupling state (unusual).
3. Monodirectional linear coupling control: L_N -> P_D -> E_F
4. Bidirectional linear coupling control: L_N -> P_D -> L_Omy -> E_F

Orthogonal

1. Orthogonal coupling -> substrate control (mono- or bidirectional)
2. Orthogonal substrate -> coupling control (mono- or bidirectional)

Diametral

1. Diametral coupling -> substrate ->> control (mono- or bidirectional)
2. Diametral substrate -> coupling ->> control (mono- or bidirectional)

SUIT library

CI + CI&CII + CII diametral

Respiratory states: CI_L -> CI_P -> CI&II_P -> CI&II_E -> CII_E -> ROX

• GM+D+S+U+Rot Innsbruck-Copenhagen protocol: GM+D+c+S+U+Rot ::Cyt c: added after ADP; cyt c added after S; ADP conc: _ _; ROX: Ama (Boushel 2007 Diabetologia)

• PMG+D+S+U+Rot Brain protocol: PMG+D+c+S+U+Rot::ADP conc: in mouse brain homogenate a final conc. of 2 mM ADP in the OXPHOS state (CI&II) is saturating (Sumbalova 2011 Abstract Bordeaux); ROX: Mna&Ama; Myx

CI + CI + CI&CII + CII diamteral

• GM+D+P+S+U+Rot Innsbruck-Liege protocol 1: GM+D+c+P+S+U+Rot::Cytochrome c: early addition of c ensures comparability of all states in case of any c effect.

• PM+D+G+S+U+Rot Innsbruck-Liege protocol 2: PM+D+c+G+S+U+Rot

FAO + CI + CI&CII + CII diametral

Respiratory states: FAO_L -> FAO_P -> CI&FAO_P -> CI&II&FAO_P -> CI&II&FAO_E -> CII_E -> ROX

• OctM+D+G+S+U+Rot Innsbruck training protocol: OctM+D+G+c+S+U+Rot::Pesta 2012 Methods Mol Biol, Pesta 2011 Am J Physiol Regul Integr Comp Physiol

Respiratory states: FAO_L -> FAO_P -> CI&FAO_P -> CI&II&FAO_P -> CII_P -> CII_L -> CII_E -> ROX

• OctM+D+G+S+Rot+Omy+U Greenland-Monte Rosa protocol: OctM+D+G+S+Rot+Omy+U+c::Cyt c: added after U. The absence of a significant c-effect shows the integrity of the outer mitochondrial membrane after fibre preparation and also shows the preservation of integrity over a 90-100 min incubation in the O2k (MiR05); Oxygen: oxygen limitation of flux was preventd by maintaining oxygen levels in the respirometer above air saturation (Pesta 2012 Methods Mol Biol); ROX: Mna+Ama

References and notes

• MitoPedia: Respiratory states
• MitoPedia: Terms and abbreviations

• Gnaiger_2014_MitoPathways
• Gnaiger_2009_Int J Biochem Cell Biol
• Pesta 2012 Methods Mol Biol
• Pesta 2011 Am J Physiol Regul Integr Comp Physiol
• Sumbalova 2011 Abstract Bordeaux