Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP

ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [gamma-P-32]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.

Automated summary

ATPase assays are commonly used for characterizing purified ATPases. This radioactive [gamma-P-32]-ATP-based approach described here can be highly sensitive and is suitable for proteins with low ATPase activity or low purification yields. It can be used for various applications including substrate identification, determining the effect of mutations on ATPase activity, and testing ATPase inhibitors.