Hi Utsav,

you can do a malachite green assay that is baed on ammoniummolydate that binds inorganic phosphate (that is released upon hydrolysis of ATP by the ATPase). In the phsphate-bound form and in complex with malachite green you can see a shift from yellow to green and quantify the reaction by its absorption at 660 nm.  You can stop the reaction with the addition of sodium citrate.

pretty easy and reliable. 

you can purchase a kit from a company or mix it yourself - you can find many protocols online. 

good luck, Janine

Dear Janine,

Thanks a lot for the input. It was really helpful. I got a paper on this as well after searching with your inputs. " ATPase Activity of p97-Valosin-containing Protein (VCP)" JBC, Vol. 278, No. 6, Issue of February 7, pp. 3648–3655, 2003. Will get back to you if I need anymore help on that. 

Thanks!

Best,

Utsav

There are a few ATPase assays known in the literature. In one set of assays the inorganic phosphate (Pi) liberated in the reaction is determined colorometrically. In another set of assays the liberated ADP is measured in a coupled enzyme assay where the ADP is converted back to ATP and the change of concentration of some other chemical compound (for instance NADH) is detected in time. The advantage of this latter assay is that the ATP concentration is kept constant in time.

You can also measure Pi concentration using UV-detection method. It is fast and simple (see attatched link).

An assay of Pi liberation is quite o.k. for determination of various kinds of ATPases in which one can use various inhibitors in order to distinguish ATPase/ADPase/AMPase activities. The details you can find in the following paper: Sabolic et al.  Am J Physiol Renal Physiol 262:F217-F228, 1992 and in other papers cited in this work. Have fun. $

I have a assay for NaK ATPase that I have been using for gills in crabs and fishes

Hi, Pi liberation is OK with membrane vesicles, all controls should be done.

If you would like to determine it in a biochemist manner, you can use Pi liberation assay via reaction with ammonium molybdate as suggested above. However, it will takes hours to setup the reaction and to obtain the results.

If you are more a biochemist/physiologist, you can use an enzym coupled assay (pyruvate Kinase+lactate deshydrogenase). ATPase activity will be estimate from consumption of NADH at 340nm. It allows to follow the activity directly on a UV spectrophotometer in a real-time manner. The exciting point is that you will see the activity of your protein immediately on the screen and you can add effectors in the cuvette if you want.

If you are more a biophysician, you can use 31P NMR to follow the hydrolysis of ATP. Pi has a peculiar signal that is easy to follow and to quantify.

Hi Utsav! You have to think about the initial substrate concentration and the product that will be released during your assay to make a choice of the most suitable method. If you are starting with millimolar ATP concentrations, and you believe that your ATPase activity will be in the micromol/mg/min range, and you can use several micrograms of protein per assay, the method of the molybdovanadate reagent (Lin and Morales, 1977) will do well as it is a quite simple one. If your initial ATP concentration is much lower (micromolar), or the specific activity or enzyme concentrations are low, the acid molybdate+malachite green assay will do better because of its higher sensitivity. In this case, be careful to use the correct malachite green and sulfuric acid concentration. If you have a good spectrophotometer, and can mix additional protein without trouble, the coupled assay with PK+LDH, where you follow NADH oxidation, will be useful since you can vary (increase sequentially, of course) either the substrate or the enzyme concentration on the cuvette to determine the best experimental condition. I'm glad to see that so many people are trying to help you! Greetings to all of you!

Hi Utsav: You can assay ATPase by using our published article: JBC, 262: 5664-5670, 1987. Also, you can check quick activity of ATPase by using a partial reaction of ATPase using K-p-NPPase activity.

Hi Utsav! I have utilized the spectrophotometric coupled assay with ATP, PK+LDH, where you follow NADH oxidation, as long as added ATP is trasnformed into ADP , We have published it in : Cell Mol Neurobiol. 2013 Jul;33(5):637-49. doi: 10.1007/s10571-013-9926-7. utilzing the assay to test the extramitochondiral FoF1-ATP sythase working as ATPase, best!

Yes, since 1925 Fiske and Subbarow published a simple colorimetric dedermination of the inorganic phosphate released from the ATPase reaction. This method is adequately modified according to the optimal conditions required by the  ATPase assay (pH, cations etc.) which should be defined

Article [Na+-ATPase in the gills of bass (Dicentrarchus labrax)]

Article Tributyltin inhibits the oligomycin-sensitive Mg-ATPase acti...

Article Response to alkyltins of two Na+-dependent ATPase activities...

Yes, however,  I have utilized many times Fiske and Subbarow method, however I  stopped using it since I realized that it needs particular attention  in the presence of  Calcium . I have made multiple exeriments without sample in the presence of increasing conc. of Ca2+ and have noticed that there can be an artifact due to the formation of apatite also in dependence of the amount of produced phosphate and pH! it is advisable to do some pilot assays without sample... best!

Thank you all for your valuable advises. I really appreciate the concern and the help. I have come up with a protocol after reading your suggestions and planning to set up the assay soon. Thanks again! Cheers!!

You can use sensitive assay for estimating released inorganic phosphate Pi. It works well in our hands. I enclose two PDFs for your information

 I agree with the coleagues!  All the methods cited can be used, depending on the sensitivity you need.

regards,

Ana

Attached you will find ATPase assay,