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

In English

Författare

Summary, in 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.

Publiceringsår

2004

Språk

Engelska

Sidor

193-202

Publikation/Tidskrift/Serie

Biochemical Journal

Volym

380

Issue

1

Dokumenttyp

Artikel i tidskrift

Förlag

Portland Press

Ämne

  • Biochemistry and Molecular Biology

Nyckelord

  • complex 1
  • alamethicin
  • NADH dehydrogenase
  • electron-transport chain
  • permeabilization
  • plant mitochondria

Status

Published

Forskningsgrupp

  • Plant Biology

ISBN/ISSN/Övrigt

  • ISSN: 0264-6021