SUIT-027 O2 ce-pce D065: Difference between revisions
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::: '''[[SUIT protocol pattern]]:''' ce1;1Dig;1M;2D;3M;4P;5G- | ::: '''[[SUIT protocol pattern]]:''' ce1;1Dig;1M;2D;3M;4P;5G- | ||
This protocol is focused on the analysis of the [[Malate anaplerotic pathway control state|malate anaplerotic pathway control state]] in [[mitochondrial preparations]] (isolated mitochondria, permeabilized cells, homogenate and permeabilized fibers). [[Malate]] alone does not support respiration if | This protocol is focused on the analysis of the [[Malate anaplerotic pathway control state|malate anaplerotic pathway control state]] in [[mitochondrial preparations]] (isolated mitochondria, permeabilized cells, homogenate, and permeabilized fibers). [[Malate]] alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP<sup>+</sup>- or NAD(P)<sup>+</sup>-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. [[PM]] or [[GM]]). | ||
__TOC__ | __TOC__ | ||
ย Communicated byย [[Iglesias-Gonzalez J]], [[Cardoso LH]] | ย Communicated byย [[Iglesias-Gonzalez J]], [[Cardoso LH]], [[Gnaiger E]] and [[Garcia-Souza LF]] (last update 2019-07-01) | ||
{{Template:SUIT-027 O2 ce-pce D065}} | {{Template:SUIT-027 O2 ce-pce D065}} | ||
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:::# The presence of malate anaplerotic pathway due to mitochondrial [[malic enzyme]] in the sample studied. | :::# The presence of malate anaplerotic pathway due to mitochondrial [[malic enzyme]] in the sample studied. | ||
:::# Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study [[F-pathway]] without [[N-pathway]] contribution in samples with mt malic enzyme. ย | :::# Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study [[F-pathway]] without [[N-pathway]] contribution in samples with mt malic enzyme. ย | ||
:::- This protocol does not | :::- This protocol does not analyze [[Coupling control state|coupling control]]. | ||
:::- Different [[pathway control states]] are not | :::- Different [[pathway control states]] are not analyzed in this protocol. | ||
:::- The DLP for SUIT-027 O2 ce-pce D065 is provided without the [[antimycin A]] addition step. However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by adding an Event&Mark for the antimycin A titration (6Ama) after glutamate (5G) to obtain ROX. | :::- The DLP for SUIT-027 O2 ce-pce D065 is provided without the [[antimycin A]] addition step. However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by adding an Event&Mark for the antimycin A titration (6Ama) after glutamate (5G) to obtain ROX. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-007]]: Protocol to | ::::* [[SUIT-007]]: Protocol to analyze glutamate anaplerotic pathway. | ||
::::* [[SUIT-002]]: A low concentration of malate is initially used to assess F-pathway without N-pathway contribution. Further, a higher concentration of malate is added to | ::::* [[SUIT-002]]: A low concentration of malate is initially used to assess F-pathway without N-pathway contribution. Further, a higher concentration of malate is added to analyze N-pathway. | ||
Revision as of 17:34, 1 July 2019
Description
Abbreviation: Malate anaplerosis
Reference: A - SUIT-027
SUIT number: D065_ce1;1Dig;1M;2D;3M;4P;5G
O2k-Application: O2
- MitoPedia: SUIT - Protocol for analysis of the anaplerotic pathway
- SUIT protocol pattern: ce1;1Dig;1M;2D;3M;4P;5G-
This protocol is focused on the analysis of the malate anaplerotic pathway control state in mitochondrial preparations (isolated mitochondria, permeabilized cells, homogenate, and permeabilized fibers). Malate alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP+- or NAD(P)+-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. PM or GM).
Communicated by Iglesias-Gonzalez J, Cardoso LH, Gnaiger E and Garcia-Souza LF (last update 2019-07-01)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | Dig | ce1;1Dig
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1M.05 | (ML) | ce1;1Dig;1M | ||
2D | ( MP) | ce1;1Dig;1M;2D
| ||
3M | MP | N | CI | ce1;1Dig;1M;2D;3M
|
4P | PMP | N | CI | ce1;1Dig;1M;2D;3M;4P
|
5G | PGMP | N | CI | ce1;1Dig;1M;2D;3M;4P;5G
|
6Ama | ROX | ce1;1Dig;1M;2D;3M;4P;5G;6Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- + Multiple titrations with malate provide information on:
- The presence of malate anaplerotic pathway due to mitochondrial malic enzyme in the sample studied.
- Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study F-pathway without N-pathway contribution in samples with mt malic enzyme.
- - This protocol does not analyze coupling control.
- - Different pathway control states are not analyzed in this protocol.
- - The DLP for SUIT-027 O2 ce-pce D065 is provided without the antimycin A addition step. However, it is possible to create a DLPU by adding an Event&Mark for the antimycin A titration (6Ama) after glutamate (5G) to obtain ROX.
- + Multiple titrations with malate provide information on:
Compare SUIT protocols
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry