Abstract
Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD+ or NADPH/NADP+ absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fiske CH, Subbarow Y (1925) Colorimetric determination of phosphorus. J Biol Chem 66:375–400
Baginski ES, Foa PP, Zak B (1967) Determination of phosphate: study of labile organic phosphate interference. Clin Chim Acta 15:155–158
Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95–97
Warren GB, Toon PA, Birdsall NJ, Lee AG, Metcalfe JC (1974) Reconstitution of a calcium pump using defined membrane components. Proc Natl Acad Sci U S A 71:622–626
Møller JV, Lind KE, Andersen JP (1980) Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein-protein interactions. J Biol Chem 255:1912–1920
Rayan G, Adrien V, Reffay M, Picard M, Ducruix A, Schmeetz M, Urbach W, Taulier N (2014) Surfactant bilayers maintain transmembrane protein activity. Biophys J 107:1129–1135
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sehgal, P., Olesen, C., Møller, J.V. (2016). ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3179-8_11
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
eBook Packages: Springer Protocols