More tricks: determination of ATP consumption rate and ATP/ADP ratio
Since with this methodology one can measure the ATP efflux from mitochondria mediated by the ANT, it can also be applied for measuring ATP consumption by sufficiently depolarized mitochondria. Furthermore, since the total amount of [ADP]+[ATP] remains constant in the extramitochondrial medium, the extramitochondrial ATP/ADP ratio can also be calculated. An example of this is shown in the figures below:
In the panels above, the reversibility of the ANT and estimation of extramitochondrial ATP/ADP ratio is depicted. These are reconstructed time courses of free [Mg2+], calculated from MgG fluorescence in liver mitochondria. 0.5 mM ATP and 0.5 mM ADP were added where indicated. After consumption of ADP, there is no further alteration in free [Mg2+]. Upon addition of 0.1 mM SF 6847 a progressive elevation in free [Mg2+] is observed reflecting a decrease in extramitochondrial ATP and concurrent increase in ADP. This reaction process continues until ATP is consumed. B: Time course of [ATP]t in the medium, calculated from the data in panel A. C: Time course of [ATP]t/[ADP]t in the medium, calculated from the data in panel B. Panels A, B and C are aligned on the x-axis (time, sec). Adapted from Biophys J. 2009 Mar 18;96(6):2490-504.