I would like to measure ATP level in Arabidopsis by Promega ENLITEN® ATP Assay kit. If I want to simply look at the ATP produced in mitochondria, can I grow Arabidopsis in dark then measure the ATP level?
You may use Promega kit or any other commercial tool based on the luciferine-luciferase method for measuring the overall ATP content of a given sample. However, more informative than ATP alone is the ATP/ADP ratio or, if AMP plays any role, the 'energy charge', EC, that is the ratio ([ATP] + [ADP]/2)/([ATP] + [ADP] + [AMP]).
Whatever it may be, the question is: what (and why) are you looking for? When you are mentioning mitochondria, do you mean leaf or root mitochondria? If from leaves, you cannot neglect chloroplast contribution (not mentioning other organelles and cytosol, however of less importance). In this case, you will have to extract intact mitochondria, free of chloroplasts: numerous methods are described in literature. You should block ATP synthesis or hydrolysis, by oligomycin, for example. Notice that the ATP pool and ATP/ADP ratio in leaves depend on metabolism. It will not be the same under illumination (photosynthesis and respiration interact) or in darkness (only respiration operates, but not identically to that occurring in light). Finally, more instructive may be the dynamic aspect of mitochondrial functioning. It may be followed in a continuous way, by oxygen uptake, using a Clark type electrode, and for ATP synthesis with a good and sensitive glass electrode. The later monitors the small pH increase in a slightly buffered medium resulting from the stoichiometric H+ consumption due to the reaction (Mg.ADP)- + (HPO4)2- + H+⇆ (Mg.ATP)2-: see Nishimura M., Ito K. and Chance B. (1962) Biochim. Biophys. Acta 59, 177-182.
Yaroslav, I am grateful for all these information you provided. Actually I want to investigate if a mitochondria located protein will increase the ATP production. If I just use etiolated plant leaves to perform the luciferine-luciferase assay, is it will explain the increased ATP result from the over-expressed mitochondria protein? BTW, can I use root to do this experiment? If yes, how the result will be different with the leaves? Thanks!
Hello Zhang.Can you please tell me how did you measure the ATP in mitochondria? Can we do it by isolating it?
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