Bioluminometric method for real-time detection of ATPase activity.
We have developed a real-time, simple, and sensitive method for the detection of ATP hydrolysis activity (ATPase) of apyrase (EC 3.6.1.5). The assay is based on the continuous monitoring of the ATP hydrolysis reaction using the firefly luciferase system. The method is sensitive and yields linear responses between 0.7 and 70 mU for the Solanum tuberosum apyrase. The detection limit was found to be 0.7 mU apyrase. We used the method to study the inhibitory effects of various compounds on the ATPase activity of potato apyrase, measured with 500 nM ATP. The concentrations of azide, AMP, Pi, fluoride, and ADP, which inhibit the ATPase activity by 50% (IC50), were found to be approximately 100, 0.25, 0.125, 0.04, and 0.035 mM, respectively. Under our assay conditions, vanadate inhibited about 98% of the ATPase activity of the potato apyrase at a concentration of 250 microM. The possibility of using the new method for other applications is discussed.
Karamohamed
S
S
Harvard University, Cambridge, MA, USA.
Guidotti
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D006868:Hydrolysis; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D013379:Substrate Specificity; D014638:Vanadates
D004791:Enzyme Inhibitors; D014638:Vanadates; D000255:Adenosine Triphosphate; D008156:Luciferases; D000251:Adenosine Triphosphatases; D001081:Apyrase