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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

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Author

Summary, in English

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.

Publishing year

2005

Language

English

Pages

695-704

Publication/Series

Biochemical Journal

Volume

389

Issue

Pt 3

Document type

Journal article

Publisher

Portland Press

Topic

  • Biochemistry and Molecular Biology

Keywords

  • respiratory enzyme
  • alamethicin permeabilization
  • plant cell survival
  • mitochondria
  • membrane
  • plasma
  • tonoplast

Status

Published

Research group

  • Plant Biology

ISBN/ISSN/Other

  • ISSN: 0264-6021