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Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria

Author:
  • Fredrik I Johansson
  • Agnieszka Michalecka
  • IM Moller
  • Allan Rasmusson
Publishing year: 2004
Language: English
Pages: 193-202
Publication/Series: Biochemical Journal
Volume: 380
Issue: 1
Document type: Journal article
Publisher: Portland Press Limited
Additional info: The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Biology building (Closed 2011) (011008000), Department of Cell and Organism Biology (Closed 2011.) (011002100)

Abstract english

The inner mitochondrial membrane is selectively permeable, which limits the transport of solutes and metabolites across the membrane. This constitutes a problem when intramitochondrial enzymes are studied. The channel-forming antibiotic AlaM (alamethicin) was used as a potentially less invasive method to permearbilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD(P)H dehydrogenarses as well as matrix enzymes in situ. AlaM was found to inhibit the electron-tran sport chain at the external Ca2+-dependent rotenone-insensitive NADH dehydrogenase and around complexes III and IV. Nevertheless, under optimal conditions, especially complex I-mediated NADH oxidation in AlaM-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial matrix and allowed the determination of NAD(+) requirements of malate oxidation in situ. In summary, we conclude that AlaM provides the best method for quantifying NADH dehydrogenase activities and that AlaM will prove to be an important method to study enzymes under conditions that resemble their native environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes.

Keywords

  • Biochemistry and Molecular Biology
  • complex 1
  • alamethicin
  • NADH dehydrogenase
  • electron-transport chain
  • permeabilization
  • plant mitochondria

Other

Published
  • ISSN: 0264-6021
Allan Rasmusson
E-mail: allan [dot] rasmusson [at] biol [dot] lu [dot] se

Professor

Molecular Cell Biology

+46 46 222 93 81

B-A329A

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