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Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin

Rasmusson, Allan G. LU ; Møller, Ian Max and Widell, Susanne LU (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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Please use this url to cite or link to this publication:
author
; and
organization
publishing date
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
1064-3745
1940-6029
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
2024-06-15 20:37:00
@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         = {{1064-3745}},
  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}},
}