The current method exploits the differential affinity of ADP and ATP to Mg2+ (Kd_ADP and Kd_ATP, respectively). The rate of ATP appearing in the extramitochondrial medium following 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 (MgG), using standard binding equations. The assay is designed so that the ANT is the sole mediator of changes in [Mg2+] in the extramitochondrial volume, as a result of ADP-ATP exchange. The principle is depicted in the cartoon below:
I emphasize that the novelty of the methodology outlined in this website and in the original publication is that we could convert the changes in free [Mg2+] to changes in [ATP], using standard binding equations. Thus, we developed a quantitative method for measuring ATP output from mitochondria. Since the ANT exchanges ADP for ATP in a 1:1 format, the rate of ATP efflux from polarized mitochondria is the rate of ADP-ATP exchange by the ANT.