Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin

Allan G. Rasmusson*, Ian Max Møller, Susanne Widell

*Corresponding author for this work

Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review

2 Citations (Scopus)

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 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.

Original languageEnglish
Title of host publicationPlant Mitochondria
EditorsOlivier Van Aken , Allan G. Rasmusson
Number of pages8
Place of publication New York
PublisherHumana Press
Publication date2022
Pages77-84
Chapter7
ISBN (Print)978-1-0716-1652-9
DOIs
Publication statusPublished - 2022
SeriesMethods in Molecular Biology
Volume2363
ISSN1064-3745

Keywords

  • Alamethicin
  • BY-2 cells
  • Electron transport chain
  • Mitochondria
  • Tricarboxylic acid cycle

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