Integrity Assessment of Isolated Plant Mitochondria

Allan G. Rasmusson*, Mengshu Hao, Ian Max Møller

*Corresponding author for this work

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

Abstract

The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.

Original languageEnglish
Title of host publicationPlant Mitochondria
EditorsOlivier Van Aken, Allan G. Rasmusson
Number of pages12
Place of publicationNew York
PublisherHumana Press
Publication date2022
Pages51-62
Chapter5
ISBN (Print)978-1-0716-1652-9
DOIs
Publication statusPublished - 2022
SeriesMethods in Molecular Biology
Volume2363
ISSN1064-3745

Keywords

  • Alamethicin
  • Complex I
  • Cytochrome c
  • H transport
  • Latency
  • Membrane integrity
  • Mitochondrial quality

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