Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin

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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
Subtitle of host publicationMethods and Protocols
EditorsOlivier Van Aken, Allan g. Rasmusson
PublisherHumana Press
Number of pages8
ISBN (Electronic)978-1-0716-1653-6
ISBN (Print)978-1-0716-1652-9
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

Free keywords

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


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