Abstract
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. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases), that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides 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 (originally employed for phosphate detection in environmental analysis) to allow its use with phosphatase enzymes. In particular, 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.
Highlights
Adenosinetriphosphatases (ATPases) are enzymes that produce inorganic phosphate (Pi) by cleavage of the c-phosphate of ATP
When determining the hydrolytic activity of ATPases, acid ATP hydrolysis occurs during the time elapsed between addition of the aliquot to the coloring solution and absorbance measurement (‘‘elapsed time’’)
Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements
Summary
Adenosinetriphosphatases (ATPases) are enzymes that produce inorganic phosphate (Pi) by cleavage of the c-phosphate of ATP. Main representative members of this large family are the cationtransport ATPases, e.g. sarcoplasmic reticulum Ca-ATPase (SERCA) [1,2] and Na,K-ATPase [3,4]. These proteins couple ATP hydrolysis to the transport of ionic species against their electrochemical potential gradient. The enzymatic cycle includes initial enzyme activation triggered by cation binding, followed by ATP utilization to form a phosphorylated intermediate. Binding of counter-transported ions induces dephosphorylation of the enzyme, followed by release of the counterions during a conformational transition to the initial state [5]. Pi detection is useful to evaluate the rate of Pi production by ATPases and the related enzyme activity, an extremely important functional parameter
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