SUIT protocol library

Substrate-uncoupler-inhibitor titration (SUIT) protocols

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

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Strategy

Select a pathway control state.
Select a control pattern.
Define substrates for each pathway state.
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

CI
CII
CI&II
CIV
FAO
CGpDH

Multiple pathway control states

A. CI-CII-CIV

CI + CII
CI + CII + CIV
CI + CI&II + CII
CI + CI&II + CII + CIV

B. FAO-CI-CII-CGpDH-CIV

FAO + CI&FAO + CII + CGpDH
FAO + CI&FAO + CII + CGpDH + CIV
FAO + CI&FAO + CI&II&FAO + CII + CGpDH
FAO + CI&FAO + CI&II&FAO + CII + CGpDH + CIV

C. FAO-CI-CII-CIV

FAO + CI&FAO + CII
FAO + CI&FAO + CII + CIV
FAO + CI&FAO + CI&II&FAO + CII
FAO + CI&FAO + CI&II&FAO + CII + CIV

D. CGpDH-CI-CII-CIV

CGpDH + CI&GpDH
CGpDH + CI&GpDH + CII&GpDH
CGpDH + CI&GpDH + CII&GpDH + CIV
CGpDH + CI&GpDH + CI&II&GpDH + CII&GpDH
CGpDH + CI&GpDH + CI&II&GpDH + CII&GpDH + CIV
CI + CI&GpDH + ...
CI + CI&II + CI&II&GpDH + ...
CI&II + CI&II&GpDH + ...

E. FAO-CGpDH-CI-CII-CIV

FAO + FAO&GpDH + CI&FAO&GpDH + ...
CGpDH + FAO&GpDH + CI&FAO&GpDH + ...
FAO + FAO&GpDH + CI&II&FAO + CI&II&FAO&GpDH + ...
CGpDH + FAO&GpDH + CI&FAO&GpDH + ...
CI + CI&II + CI&II&GpDH + CI&II&FAO&GpDH + ...
...

Control patterns

Linear

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

Orthogonal

Orthogonal coupling -> substrate control (mono- or bidirectional)
Orthogonal substrate -> coupling control (mono- or bidirectional)

Diametral

Diametral coupling -> substrate ->> control (mono- or bidirectional)
Diametral substrate -> coupling ->> control (mono- or bidirectional)

SUIT library

CI + CI&II + 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&II + 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&II + 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