Integrity Assessment of Isolated Plant Mitochondria
(2022) In Methods in Molecular Biology 2363. p.51-62- Abstract
The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of... (More)
The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.
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- author
- Rasmusson, Allan G. LU ; Hao, Mengshu LU and Møller, Ian Max
- organization
- publishing date
- 2022
- type
- Chapter in Book/Report/Conference proceeding
- publication status
- published
- subject
- keywords
- Alamethicin, Complex I, Cytochrome c, H transport, Latency, Membrane integrity, Mitochondrial quality
- host publication
- Methods in Molecular Biology
- series title
- Methods in Molecular Biology
- volume
- 2363
- pages
- 12 pages
- publisher
- Humana Press
- external identifiers
-
- pmid:34545485
- scopus:85115857816
- ISSN
- 1064-3745
- 1940-6029
- DOI
- 10.1007/978-1-0716-1653-6_5
- language
- English
- LU publication?
- yes
- additional info
- Publisher Copyright: © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
- id
- 98a62f82-a55d-4cac-acc6-bf50bb691427
- date added to LUP
- 2021-10-14 11:23:58
- date last changed
- 2024-08-10 23:06:32
@inbook{98a62f82-a55d-4cac-acc6-bf50bb691427,
abstract = {{<p>The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD<sup>+</sup>, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.</p>}},
author = {{Rasmusson, Allan G. and Hao, Mengshu and Møller, Ian Max}},
booktitle = {{Methods in Molecular Biology}},
issn = {{1064-3745}},
keywords = {{Alamethicin; Complex I; Cytochrome c; H transport; Latency; Membrane integrity; Mitochondrial quality}},
language = {{eng}},
pages = {{51--62}},
publisher = {{Humana Press}},
series = {{Methods in Molecular Biology}},
title = {{Integrity Assessment of Isolated Plant Mitochondria}},
url = {{http://dx.doi.org/10.1007/978-1-0716-1653-6_5}},
doi = {{10.1007/978-1-0716-1653-6_5}},
volume = {{2363}},
year = {{2022}},
}