Measurement of ATPase Activity of Valosin-containing Protein/p97
Kruthi Suvarna,
Kaori Honda,
Makoto Muroi,
Yasumitsu Kondoh,
Hiroyuki Osada,
Nobumoto Watanabe
Affiliations
Kruthi Suvarna
Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, JapanTokyo Medical Dental University, Yushima, Tokyo, 113-8510, Japan
Kaori Honda
Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, JapanChemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
Makoto Muroi
Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
Yasumitsu Kondoh
Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
Hiroyuki Osada
Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, JapanRIKEN-Max Planck Joint Research Division, RIKEN CSRS, Saitama, 351-0198, Japan
Nobumoto Watanabe
Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, JapanTokyo Medical Dental University, Yushima, Tokyo, 113-8510, Japan, RIKEN-Max Planck Joint Research Division, RIKEN CSRS, Saitama, 351-0198, Japan
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.
