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Publikationstyp: Zeitschriftenaufsatz
Dokumenttyp: Originalarbeit

Jahr: 2020

AutorInnen: Kreiter, J; Beitz, E; Pohl, EE

Titel: A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes.

Quelle: Biomolecules. 2020; 10(5):685



Autor/innen der Vetmeduni Vienna:

Kreiter Jürgen
Pohl Elena

Beteiligte Vetmed-Organisationseinheiten
Institut für Physiologie, Pathophysiologie und Biophysik, Abteilung für Physiologie und Biophysik


Zugehörige(s) Projekt(e): Proton leak through Adenine Nucleotide Translocase


Abstract:
Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.


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