title: A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases).
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.
authors.affiliation: Department of Chemistry Ugo Schiff, University of Florence, Sesto Fiorentino, Italy. gianluca.bartolommei@unifi.it
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bartolommei
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Moncelli
authors.forename: Francesco
authors.initials: F
authors.lastname: Tadini-Buoninsegni
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000737:chemistry*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002412:Cations; D006868:Hydrolysis; D008982:Molybdenum / Q000737:chemistry; D018482:Muscle, Skeletal / Q000201:enzymology; D010710:Phosphates / Q000737:chemistry; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000737:chemistry
chemical_list: D002412:Cations; D010710:Phosphates; D008982:Molybdenum; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D001205:Ascorbic Acid