I am using 4 mM of ATP to start ATP hydrolysis for my V-ATPase Activity Assay and I have been getting conflicting results. Could this be because I have not been adding magnesium to activate ATP? I'm wondering if anyone knew how much of a difference it would make if I added magnesium to my ATP? Could I be getting conflicting results due to something other than not adding magnesium? Thank you!
Dear Lucy,
Magnesium ions are required for most enzymes acting on ATP and this includes V-ATPase. In general the substrate is Mg-ATP rather than ATP, the latter often being inhibitory. You can find more info at https://www.brenda-enzymes.org/all_enzymes.php?ecno=3.6.3.14&table=Metals_Ions#TAB.
Attached are some alternative assays of ATPase based on running the assay as a first order reaction monitoring the ATP depletion continuously with the firefly luciferase reaction. I can provide more detailed technical support if you are interested.
Best regards,
Arne
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