title: Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.
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 Mg(2+) with the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgGr)™, exploiting the differential affinity of ADP and ATP for Mg(2+). In particular, cells are permeabilized with digitonin in the presence of BeF3(-) 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 Mg(2+) 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.
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address: chinopoulos.christos@eok.sote.hu.
authors.forename: Christos
authors.initials: C
authors.lastname: Chinopoulos
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Kiss
authors.affiliation: Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA.
authors.forename: Hibiki
authors.initials: H
authors.lastname: Kawamata
authors.affiliation: Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA.
authors.forename: Anatoly A
authors.initials: AA
authors.lastname: Starkov
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001608:Beryllium / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D002138:Calibration; D019069:Cell Respiration; D002478:Cells, Cultured; D005459:Fluorides / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D014638:Vanadates / Q000737:chemistry; D014966:Xanthenes / Q000378:metabolism
chemical_list: D014966:Xanthenes; C453498:magnesium green; D014638:Vanadates; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001608:Beryllium; D005459:Fluorides; D010100:Oxygen; D005998:Glycine
keywords: ADP–ATP exchange; Adenine nucleotide carrier; Adenine nucleotide translocase; Bioenergetics; Oxidative phosphorylation; Systems biology