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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Logan DC (2010) Mitochondrial fusion, division and positioning in plants. Biochem Soc Trans 38:789–795. https://doi.org/10.1042/Bst0380789
Douce R, Bourguignon J, Neuburger M, Rébeillé F (2001) The glycine decarboxylase system: a fascinating complex. Trends Plant Sci 6:167–176
Wedding RT, O'Brien CE, Kline K (1994) Oligomerization and the affinity of maize phosphoenolpyruvate carboxylase for its substrate. Plant Physiol 104:613–616
Pierre JN, Prieto JL, Gadal P, Vidal J (2004) In situ C4 phosphoenolpyruvate carboxylase activity and kinetic properties in isolated Digitaria sanguinalis mesophyll cells. Photosynth Res 79:349–355. https://doi.org/10.1023/B:PRES.0000017179.31351.f0
Averet N, Fitton V, Bunoust O, Rigoulet M, Guerin B (1998) Yeast mitochondrial metabolism: from in vitro to in situ quantitative study. Mol Cell Biochem 184:67–79. https://doi.org/10.1023/A:1006830810440
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965–976. https://doi.org/10.1038/nprot.2008.61
Salabei JK, Gibb AA, Hill BG (2014) Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9:421–438. https://doi.org/10.1038/nprot.2014.018
Leitgeb B, Szekeres A, Manczinger L, Vagvolgyi C, Kredics L (2007) The history of alamethicin: a review of the most extensively studied peptaibol. Chem Biodivers 4:1027–1051. https://doi.org/10.1002/cbdv.200790095
Cafiso DS (1994) Alamethicin: a peptide model for voltage gating and protein-membrane interactions. Annu Rev Biophys Biomol Struct 23:141–165
Aidemark M, Andersson CJ, Rasmusson AG, Widell S (2009) Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells. BMC Plant Biol 9:27
Matic S, Geisler DA, Møller IM, Widell S, Rasmusson AG (2005) Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. Cv bright yellow) suspension cells. Biochem J 389:695–704
Havelund JF (2014) The plant mitochondrial proteome and its oxidative stress response. PhD thesis, PhD thesis, Aarhus University, Aarhus, DK
Degenkolb T, Dieckmann R, Nielsen KF, Grafenhan T, Theis C, Zafari D, Chaverri P, Ismaiel A, Bruckner H, von Dohren H, Thrane U, Petrini O, Samuels GJ (2008) The Trichoderma brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins. Mycol Prog 7:177–219. https://doi.org/10.1007/s11557-008-0563-3
Aidemark M, Tjellström H, Sandelius AS, Stålbrand H, Andreasson E, Rasmusson AG, Widell S (2010) Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells. BMC Plant Biol 10:274. https://doi.org/10.1111/j.1365-3040.2011.02337.x
Askerlund P, Larsson C, Widell S, Møller IM (1987) NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences. Physiol Plant 71:9–19
Jacoby RP, Millar AH, Taylor NL (2015) Assessment of respiration in isolated plant mitochondria using Clark-type electrodes. In: Whelan J, Murcha MW (eds) Plant mitochondria; methods and protocols, Methods in molecular biology, vol 1305. Springer, New York, pp 165–186
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rasmusson, A.G., Møller, I.M., Widell, S. (2022). Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin. In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1653-6_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1652-9
Online ISBN: 978-1-0716-1653-6
eBook Packages: Springer Protocols