title: A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.
abstract: A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.
authors.forename: Christos
authors.initials: C
authors.lastname: Chinopoulos
authors.forename: Szilvia
authors.initials: S
authors.lastname: Vajda
authors.forename: László
authors.initials: L
authors.lastname: Csanády
authors.forename: Miklós
authors.initials: M
authors.lastname: Mándi
authors.forename: Katalin
authors.initials: K
authors.lastname: Mathe
authors.forename: Vera
authors.initials: V
authors.lastname: Adam-Vizi
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005453:Fluorescence; D005998:Glycine / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D010101:Oxygen Consumption; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000378:metabolism; D014966:Xanthenes
chemical_list: D014966:Xanthenes; C453498:magnesium green; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D003576:Electron Transport Complex IV; D008274:Magnesium; D005998:Glycine