Contact: Dr. Christos Chinopoulos MD, PhD chinopoulos.christos@eok.sote.hu
Dependence of ANT activity on Mg2+


As you may have guessed, the rate of ADP-ATP exchange mediated by the ANT depends on Mg2+ concentration. For those who did not figure this out yet, here is a simple explanation: The ANT transports the free forms of ADP and ATP, unbound to Mg2+ (unlike the F0-F1 ATP synthase that uses the Mg-bound ADP and ATP forms). Thus, if there is Mg2+ in the medium, it will 'compete' with the ANT for ADP or ATP. The more Mg2+ there is in the medium, the stronger the competition. Some people have experienced this from a different experimental setting: when one measures mitochondrial membrane potential (i.e. with safranin O) and ADP is added, there is a small depolarization. Depending on the amount of the ADP added, when it will be all converted to ATP, there will be a repolarization almost to baseline values. If this experiment is done in the presence or absence of Mg2+, it is obvious that in the absence of Mg2+, the ADP-induced repolarization comes faster. This is because in the presence of Mg2+, the ANT is exchanging adenine nucleotides at a slower rate. This phenomenon can be seen below:
In the experiment shown above, ATP appearing in the medium is estimated using the exact same mitochondria and conditions, but variating the concentration of ADP added to the buffer. By decreasing the amount of ADP by 0.5 mM at a time (black 2 mM ADP -> red 1.5 mM ADP-> green 1.0 mM ADP-> orange trace 0.5 mM ADP) the rate of ATP appearing in the medium increases. However, the 'price' that one has to pay when using low amounts of Mg2+ or high amounts of ADP in order to obtain a high ADP-ATP exchange rate, is a decreased signal-to-noise ratio (hairy traces).


The above effect of Mg2+ on the ADP-ATP exchange rate (thus, the ANT activity) has been originally reported by Klingenberg and Pfaff, in Regulation of Metabolic Processes in Mitochondria, (edited by JM Tager, S Papa, E Quagliariello and EC Slatter). BBA Library, Vol 7. Elsevier, 1966, page 180). This is a book (but who reads books, right?). This phenomenon has been also mentioned in European J Biochem (now FEBS Journal), 1968, vol 6, pages 66-79, by Pfaff and Klingenberg.
In the experiment shown above, ATP appearing in the medium is measured using the exact same mitochondria and conditions, but variating the concentration of Mg2+ in the buffer. By increasing the amount of Mg2+ by 0.25 mM at a time (black 0.25 mM MgCl2 -> red 0.5 mM MgCl2 -> green 0.75 mM MgCl2 -> orange trace 1 mM MgCl2) the rate of ATP appearing in the medium decreases.

By the same token, the same phenomenon occurs if one variates the amount of ADP added in the medium, but keeping the concentration of total Mg2+ constant. This can be seen below: