Hi All,
I've been looking into a protein whose overexpression (which occur naturally in some cancers) leads to enhanced proliferation. i was able to detect in addition in shorter doubling time an increase in ATP concentration (per cell) of between 50-400% (depending on the Kit used and conditions). as far as i know ATP concentration are tightly regulated in cells, enhanced production should not lead to such increase in concentration. has any one encountered similar phenotypes? would this more likely be a result of increased production (Mitochondrial and Glycolysis)? or of impaired homeostasis? what would be a good method to distinguish between the two?
thank you and best regards,
Gal
ATP content in cells can be turned over within seconds to minutes at most (which is what you see when you block aerobic ATP production with e.g. cyanide and glycolysis with iodocacetate), so it is likely not enhanced ATP production but altered homeostasis that underlies the elevated ATP levels. We have long ago seen that in hepatocytes from hypoxia adapted fish (see attachment), but there is for sure some newer literature out.
I agree with Gerhard Krumschabel that rapid sampling and quenching is essential to obtain reliable data on intracellular ATP concentrations. Additionally, you may want to measure the (adenylate) energy charge, which gives a more robust indication of the energetic status of the cells than 'just' an ATP measurement. This, of course, does require the additional measurement of AMP and ADP concentrations.
The idea to measure adenylate energy charge is good. This parameter, EC=(ATP+0.5xADP)/(ATP+ADP+AMP) is generally around 0.95. I published an assay of EC in Methods in Enzymology many years ago based on the luciferase reaction, but I also have updated instructions on how to do it. Contact me, [email protected], and I will give you free of charge support.
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