Protocol for ATPase Assay? i.e. radioactive vs non-radioactive

Dr Engelbert Buxbaum engelbert_buxbaum at web.de
Wed Nov 13 06:49:42 EST 2002


dustjohn at hotmail.com wrote:

> 
> I am in need of a decent protocol to measure ATP hydrolysis in solution.
>  One way to do it is to hydrolyze radioactive ATP (cleave off the
> gamma-32P), use charcoal and TCA to remove protein/ATP/ADP and then
> scintillation count an aliquot of the supernatant.  However this method
> is not the greatest because there are high background levels of 32PPi in
> the supernatant if the ATP solution is not pure.  Does anybody have any
> suggestions as to what I should do?  Are there any relatively cheap
> non-radioactive ATPase assays?  Any ideas of a good positive control
> (I'm working with a Ser/Thr protein kinase so any enzyme along this line
> would be fantastic).


ATPase assays can be performed non-radioactively, either by messuring
released phosphate (Fiske-Subbarow, with or without malachite green
amplification) or by coupled spectrophotometric assay for ADP with
pyruvate kinase and lactate dehydrogenase. Molecular Probes
(www.probes.com) sells a fluorimetric assay kit for phosphate, but I
have not used it.

In general, non-radioactive assays are much less sensitive than the
radioactive ones (coupled < Fiske-Subbarow < malachite green). This is
particularly important for enzyme kinetics, were it can be necessary to
work with nM concentrations of ATP, which none of the 3 methods is able
to do.

In those cases (or when enzyme is limiting) the easiest, quickest and
most sensitive assay is to convert the released phosphate into
molybdato-phosphoric acid and to extract with organic solvent.
Radioactivity is counted in the organic phase. Extraction efficiency is
better than 98%. 33-P may be used instead of 32-P (longer half life
periode, no screening required).

I have used this assay with 3 different ATPases (Na/K-ATPase, Mdr1 and
Hsc70). On a good day I have performed some 400 measurements (and then
needed a week to interprete the results). See for example Eur. J.
Biochem. 265 (1999) 64-70



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