Bioluminometric method for real-time detection of ATPase activity.

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Apyrase (EC 3.6.1.5) catalyzes ATP hydrolysis, a crucial reaction in cellular energy metabolism.
• Accurate measurement of ATPase activity is essential for understanding enzyme kinetics and inhibitor screening.
• Existing methods may lack sensitivity, real-time monitoring, or simplicity.

Purpose of the Study:

• To develop a novel, real-time, sensitive, and simple assay for quantifying apyrase ATPase activity.
• To characterize the kinetic properties of Solanum tuberosum apyrase using the developed assay.
• To investigate the inhibitory effects of various compounds on potato apyrase activity.

Main Methods:

• Utilized a firefly luciferase system for continuous monitoring of ATP hydrolysis.
• Established a linear response range for Solanum tuberosum apyrase from 0.7 to 70 mU.
• Determined IC50 values for inhibitors including azide, AMP, Pi, fluoride, ADP, and vanadate.

Main Results:

• The assay demonstrated high sensitivity with a detection limit of 0.7 mU apyrase.
• Linearity was observed between 0.7 and 70 mU of Solanum tuberosum apyrase.
• IC50 values for inhibitors were determined, with vanadate showing significant inhibition (98% at 250 microM).

Conclusions:

• The developed luciferase-based assay provides a robust and sensitive method for measuring apyrase ATPase activity.
• The assay is suitable for kinetic studies and screening of enzyme inhibitors.
• The method's simplicity and sensitivity suggest potential for broader applications in biochemical research.