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

EK khatipovNO-SPAM at NO-SPAMuchicago.edu
Wed Nov 20 13:40:28 EST 2002


My newsreader acts weirdly, so I haven't been able to read other posts in
this thread. As others already recommended, a coupled enzyme assay with
pyruvate kinase/lactate dehydrogenase might be better/easier in some cases
than the GS assay I recommended before. I am sure there are PK/LDH kits
available now on the market. However, it requires that you have a
spectrophotometer that can assay kinetics, or the one equipped with a
plotter so that you could calculate the slopes yourself. The principle of
the PK/LDH system is as follows.

ADP + phosphoenol pyruvate =(PK)= ATP + pyruvate
pyruvate + NADH =(LDH)= lactate + NAD
OD340 of NADH (molar extinction coefficient 6200) is measured
spectrophotometrically. However, the sensitivity and accuracy of the assay
is not very good because you measure depletion of NADH rather than
formation. Thus, for very weak ATPase activities you could try GS assay, or
the assays that measure phosphate recommended by others. However, I remember
that the biggest concern of the phosphate-based methods is phosphate
contamination of water, reagents and glassware, so using very pure reagents
should be used.
Emir

"EK" <khatipovNO-SPAM at NO-SPAMuchicago.edu> wrote in message
news:T3BC9.124$P4.20472 at news.uchicago.edu...
> 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
> synthetase).
> 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.
>
>
>
> Emir Khatipov
>
> 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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