SUIT protocol library: Difference between revisions
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=== Define substrates === | === Define substrates === | ||
[[Substrate control state]]s are defined for each pathway control state ([[Complex I-linked substrate state |CI-linked]], [[Complex II-linked substrate state |CII-linked]], [[Complex I&II-linked substrate state |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. | [[Substrate control state]]s are defined for each pathway control state ([[Complex I-linked substrate state |CI-linked]], [[Complex II-linked substrate state |CII-linked]], [[Complex I&II-linked substrate state |CI<small>&</small>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, .. | * CI: PM, GM, PGM, M, .. | ||
* CII: S(Rot), S, SM, .. | * CII: S(Rot), S, SM, .. | ||
* CI&II: PS, GS, PMS, GMS, PGMS, .. | * CI<small>&</small>II: PS, GS, PMS, GMS, PGMS, .. | ||
* CIV: AsTM | * CIV: AsTM | ||
* FAO: Oct, Pal, ... | * FAO: Oct, Pal, ... |
Revision as of 19:33, 14 January 2016
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
- 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)
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 -> LT).
- Linear substrate control: at a single coupling state (unusual).
- Monodirectional linear coupling control: LN -> PD -> EF
- Bidirectional linear coupling control: LN -> PD -> LOmy -> EF
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: CIL -> CIP -> CI&IIP -> CI&IIE -> CIIE -> 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: FAOL -> FAOP -> CI&FAOP -> CI&II&FAOP -> CI&II&FAOE -> CIIE -> 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: FAOL -> FAOP -> CI&FAOP -> CI&II&FAOP -> CIIP -> CIIL -> CIIE -> 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