A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.
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.
Chinopoulos
Christos
C
Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.
Vajda
Szilvia
S
Csanády
László
L
Mándi
Miklós
M
Mathe
Katalin
K
Adam-Vizi
Vera
V
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
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