Difference between revisions of "SUIT-007"

Latest revision as of 11:30, 3 June 2020

high-resolution terminology - matching measurements at high-resolution

SUIT-007

Description

Reference: A: Glutamate anaplerotic pathway

SUIT protocol pattern: 1G;2D;2c;3M;4U;5Ama

The SUIT-007 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample, when glutamate alone is added, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyse the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and Electron transfer pathway coupling control states.

Communicated by Cardoso LH, Komlodi T, Doerrier C and Gnaiger E (last update 2019-02-25)

Specific SUIT protocols

SUIT-007 O2 ce-pce D030 for permeabilized cells

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1G	G_L	(N)	CI	1G N_L if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	G_P	(N)	CI	1G;2D N_P if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	Gc_P	(N)	CI	1G;2D;2c Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). 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 (OXPHOS capacity P with saturating [ADP]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M	GM_P	N	CI	1G;2D;2c;3M NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4U	GM_E	N	CI	1G;2D;2c;3M;4U NADH-linked substrates (type N-pathway to Q). 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.
5Ama	ROX			1G;2D;2c;3M;4U;5Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

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

Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis.

+ The use of glutamate alone allows to analyse the glutamate-anaplerotic pathway control state.

+ Short duration of the experiment.

+ Glutamate is easier to prepare compared to pyruvate.

+ CIV activity can be measured after this protocol.

- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity.

- This protocol does not address questions regarding the convergence of pathways at the Q-junction.

- Addition of cytochrome c after glutamate alone (1G;2D;2c) is not as appropriated to evaluate integrity of the mitochondrial external membrane as it would be with GM. However, with addition of cytochrome c only after malate (e.g. 1G;2D;3M;3c), it would not be possible to compare G_P and GM_P if there are damages to the mitochondrial external membrane, therefore it is suggested to add cytochrome c after ADP (1G;2D;2c).

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

SUIT-006: Coupling-control protocol, can be performed with GM.

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=N(GM)
+|abbr=[[Glutamate-anaplerotic pathway control state|Glutamate anaplerosis]]
-|description=[[File:1G;2D;3M;4U;5Ama.png|300px]]
+|description=[[File:1G;2D;3M;4U-.png|300px]]
-|info='''A:''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-007.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-007.pdf Versions]
+|info='''A:''' '''[[Glutamate-anaplerotic pathway control state|Glutamate anaplerotic pathway]]'''
 }}
-::: '''[[Categories of SUIT protocols |SUIT-category]]:''' N(GM)
+::: '''[[SUIT protocol pattern]]:''' 1G;2D;2c;3M;4U;5Ama
-::: '''[[SUIT protocol pattern]]:''' diametral 1G;2D;2c;3M;4U;5Ama
+The SUIT-007 is focused on the [[Glutamate-anaplerotic pathway control state|glutamate anaplerotic pathway]]. If [[Glutamate dehydrogenase|glutamate dehydrogenase]] is present in the sample, when glutamate alone is added, it will be converted to α-ketoglutarate in an [[Anaplerosis|anaplerotic reaction]], supporting respiration. After malate is added, it is possible to analyse the [[NADH Electron transfer-pathway state]] with glutamate and malate as substrates in the [[OXPHOS]] and [[Electron transfer pathway]] [[Coupling control state|coupling control states]].
 __TOC__
-  Communicated by [[Cardoso LH]] [[Doerrier C]] and [[Gnaiger E]] (last update 2019-01-21)
+  Communicated by [[Cardoso LH]], [[Komlodi T]], [[Doerrier C]] and [[Gnaiger E]] (last update 2019-02-25)
-== DLP applications ==
-[[File:Ce1;1Dig;1G;2D;2c;3M;4U-.png|350px]]
-::::* [[SUIT-007 O2 pce D030]] for permeabilized cells
-== References ==
+== Specific SUIT protocols ==
-{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-007]]
+[[File:Ce1;1Dig;1G;2D;2c;3M;4U;5Ama.png|400px]]
-|?Was published in year=Year
+[[File:D030_O2_traces.png|500px]]
-|?Has title=Reference
+::::* [[SUIT-007 O2 ce-pce D030]] for permeabilized cells
-|?Mammal and model=Organism
-|?Tissue and cell=Tissue;cell
-|format=broadtable
-|limit=5000
-|offset=0
-|sort=Was published in year
-|order=descending
-}}
 {{Template:SUIT-007}}
 == Strengths and limitations ==
-:::* Protocol focused on analysis of glutamate dehydrogenase anaplerotic pathway/glutaminolysis.
+:::* Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis.
-:::+ The use of glutamate alone allows to analyse the glutamate dehydrogenase anaplerotic pathway control state.
+:::+ The use of glutamate alone allows to analyse the glutamate-anaplerotic pathway control state.
 :::+ Short duration of the experiment.
 :::+ Glutamate is easier to prepare compared to pyruvate.
@@ Line 37: / Line 26: @@
 :::- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity.
 :::- This protocol does not address questions regarding the convergence of pathways at the Q-junction.
+:::- Addition of cytochrome ''c'' after glutamate alone (1G;2D;2c) is not as appropriated to evaluate integrity of the mitochondrial external membrane as it would be with GM. However, with addition of cytochrome ''c'' only after malate (e.g. 1G;2D;3M;3c), it would not be possible to compare G<sub>''P''</sub> and GM<sub>''P''</sub> if there are damages to the mitochondrial external membrane, therefore it is suggested to add cytochrome ''c'' after ADP (1G;2D;2c).
 :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
 == Compare SUIT protocols ==
+::::* [[SUIT-006]]: [[Coupling-control protocol]], can be performed with GM.
+== References ==
+{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-007]]
+|?Was published in year=Year
+|?Has title=Reference
+|?Mammal and model=Organism
+|?Tissue and cell=Tissue;cell
+|format=broadtable
+|limit=5000
+|offset=0
+|sort=Was published in year
+|order=descending
+}}
 {{MitoPedia concepts