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Updated: Nov 15, 2025

Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Measurement of ATPase Activity of Valosin-containing Protein/p97
Kruthi Suvarna1,2, Kaori Honda1,3, Makoto Muroi3
1Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, Japan.
Abstract:
Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ATPases are enzymes that hydrolyze ATP in a reaction resulting the release of an inorganic phosphate. This reaction can be measured using several methods, such as colorimetric, fluorescence, and radiometric assays, in addition to the bioluminescence assay mentioned here. Since the remaining ATP level after the reaction was detected using a luciferase assay, the luminescent signal indicates the ATPase activity inversely. This protocol is sensitive, rapid, and can be used for high-throughput screening assays to study the effect of compounds on ATPase function.
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