Hydrolytic activity of adenosinetriphosphatases (ATPases) measured by a new experimental method

The detection of small amounts (nanomoles) of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Cation transporting adenosinetriphosphatases (ATPases) are phosphatases that produce inorganic phosphate by cleavage of the gamma-phosphate of ATP. These membrane transporters have many physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, as well as to provide useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate to allow its use with phosphatase enzymes. The method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening. This work was supported by Ente Cassa di Risparmio di Firenze (2009.0749) and the Italian Ministry of Education, University and Research (PRIN Project 20083YM37E).