Protocol for ATPase Assay? i.e. radioactive vs non-radioactive
khatipovNO-SPAM at NO-SPAMuchicago.edu
Tue Nov 19 19:56:00 EST 2002
I think you could look in the literature for a coupled enzyme assay that
would equimolarly measure the formation of ADP. I don't remember exactly
what enzymes are generally used for this particular purpose, but I could
suggest using e.g. glutamine synthetase (GS) transferase reaction:
L-glutamine + NH4OH + ADP + Pi = gamma-glutamyl hydroxamic acid + NH3 + ATP.
Fe(III) forms coloured (brown) complexes with the hydroxamate formed in the
reaction under acidic conditions. The complexes absorb light at 540nm.
I would suggest the following protocol:
Make the substrate solution: glutamine,50m?; hydroxylamine-HCl,100mM;
NaH2PO4,50mM; Tris-HCl,50mM (or other buffer if desired); MnCl2? 6H2O,
0.25mM (required for GS activity); GS enzyme (check if ATPase negative
prep) - quantity should be determined experimentally to be
non-rate-limiting. pH to neutral (within the pH range of the glutamine
Add 1 ml of your solution where ATP is hydrolized to 1 ml of the above
substrate solution. Run the reaction for several minutes (may require
prolonged incubation as well). Stop the reaction by adding 1 ml of the stop
solution containing FeCl3 x 7H2O, 18g; trifluoroacetic acid, 10 g;
HCl(concentrated), 12mll, water up to 250ml.
Measure OD540. Compare with the standard curve prepared using pure
gamma-glutamine hydroxamate. Determine the rate.
I don't think that the fact that ATP is formed in the reaction should
matter, because as long as the rate of hydrolysis is slower that the rate of
the glutamine synthetase reaction, formation of the hydroxamate will
equimolarly reflect the rate of ADP formation (=ATP hydrolysis ). However,
you would not be able to use this method to determine affinity of your
ATPase for ATP.
For reference to GS reaction see, e.g., the paper of Stadtman PMID: 2868385
I don't know if anybody ever used such a system to measure ADP, but I
believe it should work, unless the components of the substrate solution are
inhibiting for the ATP hydrolysis. It might be not the prettiest system, but
it allows you to avoid messing up with radioactivity. And it's cheap.
Let me know what you think.
University of Chicago
"Kyle Legate" <legatek at mcmail.cis.mcmaster.ca> wrote in message
news:Pine.SOL.4.33.0211171115510.3861-100000 at mcmail.cis.mcmaster.ca...
> On 12 Nov 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).
> > Thanks for your help!
> Gamma labelled ATP, TLC assay on PEI cellulose, Phosphorimager analysis.
> Non radioactive assay if your protein is relatively active, do a search
> for malachite green. Pi binds the dye and changes its colour properties.
> ... . . . . . . . . . . . . . . .
> legatek at mcmaster.ca Kyle Legate legatek at hotmail.com
> Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
> moon musick:ritual:IDM:experimental(electronica):minimalism:glitch
> . . . . . . . . . . . . . . . ...
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