SUIT-003 O2 ce D050

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D050

Description

Abbreviation: MitoKit-CII/Plasma membrane-permeable succinate (NV118)

Reference: A: - SUIT-003

SUIT number: D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. Coupling control protocol (CCP) in living cells (ce)

SUIT protocol pattern: ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D050 protocol is designed to study CI-deficiency in living cells. Plasma membrane-permeable succinate (Snv), which is added in ROUTINE stimulates mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system. Moreover, the SUIT-003 O2 ce D050 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and ET. To study LEAK-respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET-capacity.

Communicated by Doerrier C (last update 2019-06-25)

Steps and respiratory states

Step	State	Comment - Events (E) and Marks (M)
ce1	ROUTINE	ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce1Snv	ROUTINE	ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state. MitoKit-CII/Succinate-nv (diacetoxymethyl succinate) is a plasma membrane-permeable prodrug (permeable succinate; Snv) that diffuses across the plasma membrane. Cleavage of diacetoxymethyl residues is mediated by intracellular esterases, thus releasing succinate in the intracellular space. Abliva #: 01-118-s4
(ce2Omy)	L(Omy)	ce1;ce1Snv;(ce2Omy) 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.
ce3U	E	ce1;ce1Snv;(ce2Omy);ce3U Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency. Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Rot	ROX	ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot Rotenone inhibits Complex I, thus inhibiting the TCA cycle in living cells with intact plasma membrane. In the presence of Succinate-nv (diacetoxymethyl succinate, MitoKit-CII), the intracellular release of succinate independent of the TCA cycle leads to stimulation of respiration.
ce5Ama	ROX	ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama 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.

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

The CCP-ce (SUIT-003 O2 ce D009) is a coupling control protocol for living cells. In a living cell population, the total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).

+ Reasonable duration of the experiment.

- To obtain LEAK respiration, the concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.

Compare SUIT protocols

SUIT-003 O2 ce D062 control SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE.
SUIT-003 O2 ce D060 CCP with cell permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry