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| To assess the exchange of ADP/ATP by ANT using MgG, the K<sub>d</sub> of ADP and ATP to Mg<sup>2+</sup> should be previously calculated for the pertaining experimental conditions. | | To assess the exchange of ADP/ATP by ANT using MgG, the K<sub>d</sub> of ADP and ATP to Mg<sup>2+</sup> should be previously calculated for the pertaining experimental conditions. |
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| == Use of Magnesium Green 5N hexapotassium salt to measure ADP-ATP exchange rates ==
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| {{Publication
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| |title=Chinopoulos C (2013) Use of Magnesium Green 5N hexapotassium salt to measure ADP-ATP exchange rates. Mitochondr Physiol Network 2013-02-14.
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| |info=
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| |authors=Chinopoulos C
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| |year=2013
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| |journal=MiPNet
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| |abstract=Edited by [[Fasching M]] and [[Gnaiger E]].
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| |mipnetlab=HU Budapest Chinopoulos C
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| }}
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| {{Labeling
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| |area=Respiration, Instruments;methods
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| |preparations=Isolated mitochondria
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| |topics=ATP, ADP
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| |instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
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| }}
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| === Application of technique: Online fluorescence measurement of ADP-ATP exchange ===
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| :::: <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> determination from Magnesium Green (MgG) fluorescence in the extramitochondrial volume of isolated mitochondria and conversion to [[ADP]]-[[ATP]] exchange rate: Add mitochondria to 2 ml of an incubation medium of your choice. Including the adenylate kinase inhibitor Ap5A into the medium is essential; Mg<sup>2+</sup>, which is present in the assay medium, activates adenylate kinase. [[Ap5A]] is a potent inhibitor of [[adenylate kinase]]. MgG fluorescence can be recorded in a fluorometer, using 506 nm and 530 nm excitation and emission wavelengths, respectively. MgG exhibits an extremely high quantum yield (EM<nowiki>[</nowiki>MgG]=75,000 M<sup>-1</sup>*cm<sup>-1</sup>). At the end of each experiment, minimum fluorescence (Fmin) can be measured after addition of 4 mM EDTA, followed by the recording of maximum fluorescence (Fmax) elicited by addition of 20 mM MgCl<sub>2</sub>. Free Mg<sup>2+</sup> concentration, <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub>, is calculated from the equation: <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> = (''K''<sub>d</sub>(F-Fmin)/(Fmax-F))-0.055 mM, assuming a ''K''<sub>d</sub> of 0.9 mM for the MgG-Mg<sup>2+</sup> complex. The correction term -0.055 mM is empirical, and possibly reflects chelation of other ions by EDTA that have an affinity for MgG, and alter its fluorescence. This term is needed to obtain a reliable Mg<sup>2+</sup> estimate, as determined from calibration experiments using solutions with known, stepwise increasing, Mg<sup>2+</sup> concentrations. ADP-ATP exchange rate is estimated using the recently described method by our laboratory [1], exploiting the differential affinity of ADP and ATP to Mg<sup>2+</sup>. The rate of ATP appearing in the medium following addition of ADP to energized mitochondria (or vice versa in case of de-energized mitochondria), is calculated from the measured rate of change in free extramitochondrial <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> using standard binding equations. The assay is designed such that the ANT is the sole mediator of changes in <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> in the extramitochondrial volume, as a result of ADP-ATP exchange [1]. For the calculation of <nowiki>[ATP] or [ADP]</nowiki> from <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub>, the apparent ''K''<sub>d</sub> values should be estimated as described in [1].
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| === Validation of the technique ===
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| [[Image:Mg green checkpoints.jpg| 250px| right]]
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| :::: In order be sure that the calibrated MgG signal is converted correctly to ADP-ATP exchange rate, the following 4 check-points should be sought:
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| ::::# After calibration of the MgG signal to <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub>, the initial <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> value must be equal to that added in the cuvette by the experimenter; (i.e. did you add 1 mM MgCl2 in the cuvette? Then your calibrated MgG signal must start from 1 mM free Mg2+).
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| ::::# After addition of ADP (or ATP) to the medium, the <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> value must drop to the expected level, after estimating the ''K''<sub>d</sub> of ADP (or ATP) for Mg<sup>2+</sup>; (i.e. if you estimated ''K''<sub>d</sub> of ADP for Mg<sup>2+</sup> as 0.906 mM, the addition of 2 mM ADP to 1 mM Mg<sup>2+</sup> leads to a <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> of 0.3939 mM).
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| ::::# After conversion on the calibrated <nowiki>[</nowiki>Mg<sup>2+</sup>]<sub>f</sub> to ADP, ATP level must start from “0”, if no ATP was already present in the medium, prior to adding ADP to mitochondria.
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| ::::# The ADP-ATP exchange rate must be completely sensitive to carboxyatractyloside, a specific blocker of the ANT.
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| <nowiki>[1]</nowiki> Chinopoulos C, Vajda S, Csanady L, Mandi M, Mathe K, Adam-Vizi V (2009) A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT. Biophys J 96:2490-2504.
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| === A kinetic assay of mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase ===
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| ::::» [http://antactivity.com/index.html Manual by Christos Chinopoulos]: "Follow the links appearing on the left, starting from the top. The 'core' of the method is outlined in the Buffers, chemicals, ''K''<sub>d</sub>s determination of ADP, ATP to Mg<sup>2+</sup>, and Conversion of [Mg<sup>2+</sup>]free to [ATP]. However, it is advisable to go through all of the links, if you wish to understand what you will be actually doing."
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| ::::* For ion binding constants of the ATP-system as a function of pH, temperature, and ion composition, see: Gnaiger E, Wyss M (1994) Chemical forces in the cell: Calculation for the ATP system. In: What is Controlling Life? (Gnaiger E, Gellerich FN, Wyss M, eds) Modern Trends in BioThermoKinetics 3. Innsbruck Univ Press:207-12. - [[Gnaiger 1994 BTK-207 |»Bioblast link«]]
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- high-resolution terminology - matching measurements at high-resolution
Magnesium Green
Description
Magnesium Green belongs to the extrinsic fluorophores applied for measurement of mitochondrial ATP production, based on different dissociation constants for ADP and ATP.
» MiPNet article
Abbreviation: MgG
Reference: Chinopoulos 2014 Methods Enzymol
MitoPedia methods:
Fluorometry
Magnesium GreenTM is a registered trademark and available from Thermo Fisher Scientific (former: Invitrogen) in several formulations. For measuring mitochondrial ATP production a membrane impermeant formulation must be chosen (e.g. #M3733).
Note that while its KD for magnesium is 1.0 mM, its KD for calcium is 6 µM: Magnesium Green binds stronger to calcium than to magnesium and cannot be used in the presence of significant concentrations of free calcium. Possible contaminations with transition metals should be complexed by a small (µM range) concentration of EDTA or DTPA.
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- » O2k-FluoRespirometer with Fluorescence-Sensor Blue/Filter Set MgG / CaG
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- » A kinetic assay of mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase by [Chinopoulos C]
- » Manual Fluorescent Magnesium Indicators.
Signal and output
- Signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
- Output: The focus of the output with Magnesium Green is on Type B: Flow, flux, rate.
Kd determination of ADP or ATP to Mg2+ using MgG
To assess the exchange of ADP/ATP by ANT using MgG, the Kd of ADP and ATP to Mg2+ should be previously calculated for the pertaining experimental conditions.
