Chinopoulos 2014 Methods Enzymol

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Publications in the MiPMap
Chinopoulos C, Kiss G, Kawamata H, Starkov AA (2014) Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption. Methods Enzymol 542:333-48. doi:10.1016/B978-0-12-416618-9.00017-0

Β» PMID: 24862274 Open AccessΒ»O2k-brief

Chinopoulos Christos, Kiss Gergely, Kawamata Hibiki, Starkov Anatoly A (2014) Methods Enzymol

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 Mg2+-sensitive fluorescent indicator Magnesium Green (MgGr)β„’, exploiting the differential affinity of ADP and ATP for Mg2+. In particular, cells are permeabilized with digitonin in the presence of [Formula: see text] 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 MgGrβ„’ signal into free extramitochondrial [Mg2+] 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. β€’ Keywords: ADP–ATP exchange, Adenine nucleotide carrier, Adenine nucleotide translocase, Bioenergetics, Oxidative phosphorylation, Systems biology, Magnesium Green, High-resolution respirometry, Mitochondrial membrane potential, Safranin

β€’ O2k-Network Lab: HU Budapest Chinopoulos C

Β» O2k-Fluorometry protocol for Magnesium green

Methods Enzymol

  • Galluzzi L, Kroemer G (ed) (2014) Conceptual background and bioenergetic/mitochondrial aspects of oncometabolism. Methods Enzymol 542: 509 pp. Β»Link

Cited by

  • Cardoso et al (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. doi:10.26124/bec:2021-0001


Labels: MiParea: Respiration, Instruments;methods 

Organism: Human  Tissue;cell: HEK  Preparation: Permeabilized cells  Enzyme: Adenine nucleotide translocase 

Coupling state: LEAK, OXPHOS 

HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

Safranin, MgG, O2k-brief, ATP, MitoFit 2021 MgG, MitoFit 2022 NADH, O2k-Demo, ATP 

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