[Protein-analysis] Re: Na K ATPase Protocol

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Fri Mar 26 13:19:35 EST 2010


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


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