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Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

Matic, Sandra LU ; Geisler, Daniela LU ; Moller, Ian M; Widell, Susanne LU and Rasmusson, Allan LU (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)
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author
organization
publishing date
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 Limited
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
2007-08-03 14:31:21
date last changed
2017-04-09 04:21:56
@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},
  keyword      = {respiratory enzyme,alamethicin permeabilization,plant cell survival,mitochondria,membrane,plasma,tonoplast},
  language     = {eng},
  number       = {Pt 3},
  pages        = {695--704},
  publisher    = {Portland Press Limited},
  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},
  volume       = {389},
  year         = {2005},
}