Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin
(2022) In Methods in Molecular Biology 2363. p.77-84- Abstract
We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be... (More)
We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.
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- author
- Rasmusson, Allan G. LU ; Møller, Ian Max and Widell, Susanne LU
- organization
- publishing date
- 2022
- type
- Chapter in Book/Report/Conference proceeding
- publication status
- published
- subject
- keywords
- Alamethicin, BY-2 cells, Electron transport chain, Mitochondria, Tricarboxylic acid cycle
- host publication
- Plant Mitochondria : Methods and Protocols - Methods and Protocols
- series title
- Methods in Molecular Biology
- editor
- Van Aken, Olivier and Rasmusson, Allan g.
- volume
- 2363
- pages
- 8 pages
- publisher
- Humana Press
- external identifiers
-
- pmid:34545487
- scopus:85115823387
- ISSN
- 1940-6029
- 1064-3745
- ISBN
- 978-1-0716-1653-6
- 978-1-0716-1652-9
- DOI
- 10.1007/978-1-0716-1653-6_7
- language
- English
- LU publication?
- yes
- additional info
- Publisher Copyright: © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
- id
- 1e78445d-060a-42ca-8f6c-a2bbb57e737d
- date added to LUP
- 2021-11-18 15:50:07
- date last changed
- 2024-06-29 21:39:23
@inbook{1e78445d-060a-42ca-8f6c-a2bbb57e737d,
abstract = {{<p>We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD<sup>+</sup>-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.</p>}},
author = {{Rasmusson, Allan G. and Møller, Ian Max and Widell, Susanne}},
booktitle = {{Plant Mitochondria : Methods and Protocols}},
editor = {{Van Aken, Olivier and Rasmusson, Allan g.}},
isbn = {{978-1-0716-1653-6}},
issn = {{1940-6029}},
keywords = {{Alamethicin; BY-2 cells; Electron transport chain; Mitochondria; Tricarboxylic acid cycle}},
language = {{eng}},
pages = {{77--84}},
publisher = {{Humana Press}},
series = {{Methods in Molecular Biology}},
title = {{Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin}},
url = {{http://dx.doi.org/10.1007/978-1-0716-1653-6_7}},
doi = {{10.1007/978-1-0716-1653-6_7}},
volume = {{2363}},
year = {{2022}},
}