A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells

Hibiki Kawamata, Anatoly A. Starkov, Giovanni Manfredi, C. Chinopoulos

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg2+] reported by an Mg2+-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg2+. In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF3- and sodium orthovanadate (Na3VO4) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F1Fo-ATPase due to its sensitivity to BeF3- and Na3VO4. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.

Original languageEnglish
Pages (from-to)52-57
Number of pages6
JournalAnalytical Biochemistry
Volume407
Issue number1
DOIs
Publication statusPublished - Dec 2010

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Adenosine Diphosphate
Assays
Adenosine Triphosphate
Kinetics
ATP Translocases Mitochondrial ADP
Digitonin
Mitochondria
Adenosine Triphosphatases
Ion Pumps
Phosphorylase Kinase
Citrate (si)-Synthase
Vanadates
Adenine Nucleotides
Mitochondrial Membrane Potential
Myosins
Ports and harbors
Sodium
Membranes
Proteins

Keywords

  • Adenine nucleotide carrier
  • Adenine nucleotide translocator
  • Bioenergetics
  • CC myotubes
  • Systems biology

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells. / Kawamata, Hibiki; Starkov, Anatoly A.; Manfredi, Giovanni; Chinopoulos, C.

In: Analytical Biochemistry, Vol. 407, No. 1, 12.2010, p. 52-57.

Research output: Contribution to journalArticle

Kawamata, Hibiki ; Starkov, Anatoly A. ; Manfredi, Giovanni ; Chinopoulos, C. / A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells. In: Analytical Biochemistry. 2010 ; Vol. 407, No. 1. pp. 52-57.
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