Current method; concept conceived by others
Contact: Dr. Christos Chinopoulos MD, PhD

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:
Although it may look deceptively simplistic, it is a bit more complex than that. Full details are given under 'Conversion of [Mg2+]free to [ATP]'.

The concept regarding measuring free [Mg2+] as a means to provide an index of changing ATP concentration has already been exploited by others:

- Leyssens et al (1996) from the group of Michael R Duchen have used MgG to demonstrate the relationship between mitochondrial state, ATP hydrolysis and [Mg2+]i in isolated rat cardiomyocytes.

- Even earlier, Silverman et al (1994) from the group of Michael D Stern used Mag-Indo-1 (another fluorescent dye responding to free [Mg2+]) to show that fluctuations in free [Mg2+] correlated inversely to ATP levels, associated with changes in oxygenation and contracture, in rat cardiomyocytes.

- Even before Silverman et al, Levy et al (1988) from the group of Robert E London developed fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) for use as 19F NMR indicators of free cytosolic magnesium concentration, and then Murphy et al (1989) used them as a means of following changes in [ATP] in the ischemic rat heart.

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.