Am 24.03.2010, 09:40 Uhr, schrieb RHIANNON THOMPSON
<r.thompson.1 from research.gla.ac.uk>:
> Dear Phillipp Pagel,
>> I'm not sure if you are still using this email address or even still
> work in this field, however I came across a webpage where you dicuss a
> Na K ATPase protocol. I was wondering if you still had a version of this
> protocol that you would mind forwarding to me? I'm having alot of
> trouble with a Na K ATPase assay, in my assay ouabain appears to produce
> Pi! Any help would be much appreciated.
In case you didn't notice, you are sending to a discussion group, not an
idividual. As to assays for Na/K-ATPase activity (or any other ATPase, for
that matter), you can detect the produced ADP by a coupled
spectrophotometric test (coupling with LDH and PK) or by measuring the
released Pi. The latter can be done photometrically, fluorimetrically or
(for highest sensitivity) with gamma 32- or 33P-ATP. In case of
Na/K-ATPase of course the release should be sensitive to ouabain.
W. Schoner, C. v. Ilberg, R. Kramer & W. Seubert: On the Mechanism of Na+
and K+-Stimulated Hydrolysis of Adenosine Triphosphate, Eur. J. Biochem. 1
(1967) 334-343
C.H. Fiske & Y. Subbarov: The Colorimetric Determination of Phosphorus.
J. Biol. Chem. 66 (1925) 375-400
Brune,M.; Hunter,J.L.; Corrie,J.E.T.; Webb,M.R.: Direct, Real-time
Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent
Probe and its Application to Actomyosine Subfragment 1 ATPase,
Biochemistry 33 (1994) 8262-8271
Geladopoulos,T.P.; Sotiroudis,T.G.; Evangelopoulos,A.E.: A Malachite Green
Colorimetric Assay for Protein Phosphatase Activity, Anal. Biochem. 192
(1991) 112-116
H.H. Neufeld & H.M. Levy: A Second Ouabain-Sensitive Sodium Dependent
Adenosine Triphosphatase in Brain Microsomes, J. Biol. Chem. 244 (1969)
6493-6497
