Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• ATPase assays are crucial for characterizing ATP-dependent enzymes.
• Existing methods like Malachite green and NADH-coupled assays have limitations in sensitivity.
• Low ATPase activity or purification yields can hinder protein characterization.

Purpose of the Study:

• To describe a highly sensitive radioactive assay for measuring ATPase activity.
• To enable the characterization of proteins with low ATPase activity or purification yields.
• To provide a versatile method adaptable for various applications and reconstituted systems.

Main Methods:

• Utilized radioactive [γ-³²P]-ATP as a substrate.
• Employed molybdate complex formation for phase separation of free phosphate.
• Separated free phosphate from intact ATP to quantify hydrolysis.

Main Results:

• The radioactive assay demonstrates superior sensitivity compared to Malachite green and NADH-coupled assays.
• The assay effectively measures low ATPase activity and accommodates proteins with low purification yields.
• The protocol is adaptable for studying reconstituted ATPases.

Conclusions:

• This [γ-³²P]-ATP based assay offers a sensitive and versatile tool for ATPase characterization.
• It expands the scope of investigation to include proteins previously difficult to analyze.
• The method supports diverse applications, including substrate identification and inhibitor screening.