Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption

Christos Chinopoulos, Gergely Kiss, Hibiki Kawamata, Anatoly A. Starkov

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Citations (Scopus)

Abstract

We have previously described a fluorometric method to measure ADP-ATP exchange rates in mitochondria of permeabilized cells, in which several enzymes that consume substantial amounts of ATP and other competing reactions interconverting adenine nucleotides are present. This method relies on recording changes in free extramitochondrial Mg2 + with the Mg 2 +-sensitive fluorescent indicator Magnesium Green (MgGr)™, exploiting the differential affinity of ADP and ATP for Mg2 +. In particular, cells are permeabilized with digitonin in the presence of BeF3 O and Na3VO4, inhibiting all ATP- and ADP-utilizing reactions but mitochondrial exchange of ATP with ADP catalyzed by the adenine nucleotide translocase. The rate of ATP appearing in the medium upon the addition of ADP to energized mitochondria is then calculated from the rate of change in free extramitochondrial Mg2 + using standard binding equations. Here, we describe a variant of this method involving an improved calibration step. This step minimizes errors that may be introduced during the conversion of the MgGr™ signal into free extramitochondrial [Mg 2 +] and ATP. Furthermore, we describe an approach for combining this methodology with the measurement of mitochondrial membrane potential and oxygen consumption in the same sample. The method described herein is useful for the study of malignant cells, which are known to thrive in hypoxic environments and to harbor mitochondria with profound functional alterations.

Original languageEnglish
Title of host publicationConceptual Background and Bioenergetic/Mitochondrial Aspects of Oncometabolism
PublisherAcademic Press Inc.
Pages333-348
Number of pages16
ISBN (Print)9780124166189
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Enzymology
Volume542
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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Keywords

  • ADP-ATP exchange
  • Adenine nucleotide carrier
  • Adenine nucleotide translocase
  • Bioenergetics
  • Oxidative phosphorylation
  • Systems biology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Chinopoulos, C., Kiss, G., Kawamata, H., & Starkov, A. A. (2014). Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption. In Conceptual Background and Bioenergetic/Mitochondrial Aspects of Oncometabolism (pp. 333-348). (Methods in Enzymology; Vol. 542). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-416618-9.00017-0