Instructions for using 'CORRECTION OF Mg AND CONVERSION TO ATP.xlsx' Note: you can only input data (variables) in yellow-colored boxes. Note: Calculated data appear in green-colored boxes. The excel sheet is populated with typical data; please replace them with your own. First: In column C, input the time scale of your MgG trace (in seconds). Second: In column D, input the MgG raw fluorescence data of the actual experiment where you added ADP and you wish to measure ANT activity. Third: In column B, input the cumulated amount of MgCl2 added, in mM of the experiment performed as shown in blue or orange trace of step 5 in Kds determination of ADP, ATP for Mg2+. Fourth: In column A, input the steady states of MgG raw fluorescence obtained right after each MgCl2 pulse addition, obtained from either blue or orange trace of step 5 in Kds determination of ADP, ATP for Mg2+. Fifth: In box I23, input the value of Kd_ADP (in mM), which you determined in step 6 (substeps I-VIII) in Kds determination of ADP, ATP for Mg2+. Sixth: In box I24, input the value of Kd_ATP (in mM), which you determined in step 6 (substeps I-VIII) in Kds determination of ADP, ATP for Mg2+. Seventh: In box I26, input the amount of total MgCl2 in your buffer (in mM). Eighth: In box I27, input the amount of ADP you added (in mM). Ninth: In box I31, input the concentration of free [Mg2+] calibrated from MgG fluorescence, JUST BEFORE THE ADDITION OF ADP. Tenth: In box I32, input the concentration of free [Mg2+] calibrated from MgG fluorescence, JUST AFTER THE ADDITION OF ADP.
If you have done the above correctly, columns E, F and G will be populated with the calibrated but uncorrected MgG fluorescence to free [Mg2+], the corrected free [Mg2+] in mM, and converted free [Mg2+] to [ATP], respectively. Note that data on [ATP] appearing in the medium prior to addition of ADP will be negative, and this is normal: the method does not make sense if there is no ADP added to the medium. The 'correction step' is necessary because of the slight unreliability of MgG stemming from the fact that it is a single excitation/single emission fluorescent dye. Furthermore, these data will also be depicted in the corresponding graphs to the right. By performing a linear regression of the [ATP] appearing in the medium, you can determine the rate of ANT activity.
POSSIBILITY FOR METHOD VERIFICATION: Add 1 or 2 micromolar of carboxyatractyloside (not more, otherwise you may induce permeability transition), a couple of minutes after addition of ADP to the mitochondria; this should stop the further increase in the rate of ATP appearing in the medium. If it does stop, it means that all of the ADP-ATP exchange was due to the ANT. If there is a residual rate of [ATP] appearing in the medium, it means that there are competing ADP-phosphorylating reaction(s). If you see strong downward drifts of [ATP] upon conversion of the entire amount of ADP to ATP, this indicates ATP hydrolysis; this proceeds even during the mitochondrial ATP formation step, thus your ANT activities rates are underestimated.
In order to measure ANT activity using the present methodology, one needs to add ADP to mitochondria (isolated, or in situ from permeabilized cells, or in homogenates), and record changes in MgG fluorescence. A typical experiment would look like this:
As you can see in the above graph, addition of ADP to the suspension leads to an immediate decrease in MgG fluorescence, followed by a more gradual decrease. The initial immediate decrease in MgG fluorescence is due to chelation of extramitochondrial free Mg2+ by the ADP, and the following, more gradual decrease is due to the stronger chelation of extramitochondrial free Mg2+ by the ATP exiting the mitochondria, while less and less ADP is available extramitochondrially to chelate Mg2+. Because the exchange of ADP for ATP by the ANT is a strict 1:1 stoichiometry, the rate of ATP appearing in the medium is equal to the rate of ADP-ATP exchange mediated by the ANT, thus, the ANT activity. So, first you need to calibrate MgG raw fluorescence to [Mg2+]. To do that, you can use this excel sheet, as shown below: