|Chinopoulos Christos, Vajda Szilvia, Csanady Laszlo, Mandi Miklos, Mathe Katalin, Adam-Vizi Vera (2009) A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT. Biophys J 96, 2490-504.|
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 [Mg2+] reported by the membrane-impermeable 5K+ salt of the Mg2+-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 [Mg2+] 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.
Organism: Rat Tissue;cell: Heart, Nervous system, Liver Preparation: Isolated mitochondria Enzyme: Adenine nucleotide translocase Regulation: ADP, ATP, ATP production
MgG, Safranin, ANT
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