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

Johansson, Fredrik I LU ; Michalecka, Agnieszka LU ; Moller, IM and Rasmusson, Allan LU (2004) In Biochemical Journal 380(1). p.193-202
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
complex 1, alamethicin, NADH dehydrogenase, electron-transport chain, permeabilization, plant mitochondria
in
Biochemical Journal
volume
380
issue
1
pages
193 - 202
publisher
Portland Press Limited
external identifiers
  • wos:000221735000021
  • pmid:14972026
  • scopus:2642545691
ISSN
0264-6021
DOI
10.1042/BJ20031969
language
English
LU publication?
yes
id
d9bed102-dd08-43ac-93cf-5e1b9493084e (old id 277091)
alternative location
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1224159&blobtype=pdf
date added to LUP
2007-10-23 13:13:03
date last changed
2017-09-03 04:44:39
@article{d9bed102-dd08-43ac-93cf-5e1b9493084e,
  abstract     = {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.},
  author       = {Johansson, Fredrik I and Michalecka, Agnieszka and Moller, IM and Rasmusson, Allan},
  issn         = {0264-6021},
  keyword      = {complex 1,alamethicin,NADH dehydrogenase,electron-transport chain,permeabilization,plant mitochondria},
  language     = {eng},
  number       = {1},
  pages        = {193--202},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria},
  url          = {http://dx.doi.org/10.1042/BJ20031969},
  volume       = {380},
  year         = {2004},
}