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

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Johansson, Fredrik ILund University,Lunds universitet,Biologiska institutionen,Naturvetenskapliga fakulteten,Department of Biology,Faculty of Science (author)

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

Article/chapterEnglish2004

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2004

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LIBRIS-ID:oai:lup.lub.lu.se:d9bed102-dd08-43ac-93cf-5e1b9493084e
https://lup.lub.lu.se/record/277091URI
https://doi.org/10.1042/BJ20031969DOI

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Language:English
Summary in:English

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Subject category:art swepub-publicationtype
Subject category:ref swepub-contenttype

Notes

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.

Subject headings and genre

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
complex 1
alamethicin
NADH dehydrogenase
electron-transport chain
permeabilization
plant mitochondria

Added entries (persons, corporate bodies, meetings, titles ...)

Michalecka, AgnieszkaLund University,Lunds universitet,Biologiska institutionen,Naturvetenskapliga fakulteten,Department of Biology,Faculty of Science(Swepub:lu)fysb-ami (author)
Moller, IM (author)
Rasmusson, AllanLund University,Lunds universitet,Molekylär cellbiologi,Biologiska institutionen,Naturvetenskapliga fakulteten,Molecular Cell Biology,Department of Biology,Faculty of Science(Swepub:lu)fysb-ara (author)
Biologiska institutionenNaturvetenskapliga fakulteten (creator_code:org_t)

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In:Biochemical Journal380:1, s. 193-2020264-6021

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