Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells
(2005) In Biochemical Journal 389(Pt 3). p.695-704- Abstract
- The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)(+). After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate... (More)
- The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)(+). After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD(+)). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaccous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/229404
- author
- Matic, Sandra LU ; Geisler, Daniela LU ; Moller, Ian M ; Widell, Susanne LU and Rasmusson, Allan LU
- organization
- publishing date
- 2005
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- respiratory enzyme, alamethicin permeabilization, plant cell survival, mitochondria, membrane, plasma, tonoplast
- in
- Biochemical Journal
- volume
- 389
- issue
- Pt 3
- pages
- 695 - 704
- publisher
- Portland Press
- external identifiers
-
- wos:000231186400011
- pmid:15836437
- scopus:23644447251
- ISSN
- 0264-6021
- DOI
- 10.1042/BJ20050433
- language
- English
- LU publication?
- yes
- id
- ae4de7fa-9a5c-4b41-9ea1-b6ef0de697f1 (old id 229404)
- date added to LUP
- 2016-04-01 16:38:29
- date last changed
- 2022-04-11 14:06:53
@article{ae4de7fa-9a5c-4b41-9ea1-b6ef0de697f1,
abstract = {{The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)(+). After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD(+)). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaccous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed.}},
author = {{Matic, Sandra and Geisler, Daniela and Moller, Ian M and Widell, Susanne and Rasmusson, Allan}},
issn = {{0264-6021}},
keywords = {{respiratory enzyme; alamethicin permeabilization; plant cell survival; mitochondria; membrane; plasma; tonoplast}},
language = {{eng}},
number = {{Pt 3}},
pages = {{695--704}},
publisher = {{Portland Press}},
series = {{Biochemical Journal}},
title = {{Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells}},
url = {{http://dx.doi.org/10.1042/BJ20050433}},
doi = {{10.1042/BJ20050433}},
volume = {{389}},
year = {{2005}},
}