Other methods: problems with luciferase
Contact: Dr. Christos Chinopoulos MD, PhD chinopoulos.christos@eok.sote.hu

Several other methods for determining the ATP-ADP steady-state exchange rate mediated by the ANT have been described in the past.

Some of these directly measure ADP and/or ATP by 1) thin-layer chromatography (Pedersen and Catterall, 1979), 2) high-performance liquid chromatography (Hartwick and Brown, 1975), or 3) using radioactive nucleotides (Duee and Vignais, 1969). Others employ coupled reactions which yield an end-product that can be detected either fluorimetrically by 4) monitoring the reduction of NADP+ which occurs in the presence of glucose, hexokinase, glucose-6-phosphate dehydrogenase and effluxed ATP (Williamson and Corkey, 1979), (Passarella et al., 1988) or 5) luminometrically, detecting chemiluminescence upon ATP-dependent oxidation of luciferin, catalyzed by firefly luciferase (Lemasters and Hackenbrock, 1979). Furthermore, there are techniques which employ fluorescent derivatives of nucleotides (method 6) in order to estimate the rate of release of the fluorescent molecule upon ANT-mediated exchange for ADP (Block et al., 1986).

To complement the above functional assays, the amount of ANT protein can be estimated using (method 7) a fluorescent derivative of atractyloside (Boulay et al), or, exploiting the 1:1 stoichiometry of binding of the inhibitor to the translocase, by (method 8) stepwise titration of State 3-mitochondria with cATR until respiration is completely inhibited (Schonfeld, 1990).

Among the aforementioned methodologies, only methods 4, 5 and perhaps 6 are on-line measurements, while the rest are end-point assays (although a satisfactory time resolution can be obtained using a large number of samples as detailed by (Brandolin et al., 1990). Methods 1 and 2 require expertise in specialized equipment, while method 3 requires handling of radioactive material, conditions which may render these methods cumbersome to use. However, it must be noted that these are all very sensitive assays which require small amounts of mitochondrial protein. Methods 6 and 7 rely on materials which are not commercially available, and are therefore not appealing to the broader scientific community.

Method 5 (luciferase method), if used as an on-line method, suffers from several drawbacks. First, the lack of constant proportionality between ATP concentration and luminescence, caused by product inhibition of the luciferase reaction by oxyluciferin, renders this method at best semi-quantitative. Second, for reliable estimation of the kinetics of ATP formation, the endogenous ATP concentration prior to the assay has to be determined. Third, the required use of a low ionic strength medium and room temperature restricts experiments to conditions which are nowhere near physiological. Fourth, the necessity to maintain [O2] below 50
mM, needed to avoid significant drifts in luminescence, adds extra effort to any application of this method. Nevertheless, some of the disadvantages of this method have been overcome by various modifications (Wibom et al., 1990). Overall, in my personal opinion, please do not use this method for quantitating ADP-ATP exchange rate mediated by the ANT.

Method 4 has two disadvantages. First, there are two mediating coupled reactions which may affect stoichiometry through product (glucose-6-phosphate) inhibition of hexokinase. Second, the measured signal -NADP(H) autofluorescence- precludes the simultaneous measurement of this mitochondrial parameter, plus the stimulation of mitochondrial respiration by addition of ADP affects the NAD(P)H steady state. For a comprehensive appraisal of methods to measure ATP-ADP exchange rates in mitochondria and reconstituted systems, the reader is referred to the review by Martin Klingenberg (Klingenberg, 2008).

The advantage of the method described in this webpage is that it provides kinetic information at a high acquisition rate while it does not suffer from the drawbacks described for methods 4, 5 and 6. In addition, our method can be combined with simultaneous recording of further mitochondrial parameters using other types of fluorescent dyes. As an example, MgG fluorescence has been used to report changes in intracellular [ATP] in cardiomyocytes combined with simultaneous recording of NAD(P)H autofluorescence and mitochondrial membrane potential using JC-1 (Leyssens et al., 1996). Of course, David G Nicholls would (rightlfully) give you a lecture as of why never to use JC-1 for measuring mitochondrial membrane potential