A direct colorimetric assay for Ca2+ -stimulated ATPase activity

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Measuring ATPase activity is crucial for understanding cellular energy metabolism and signaling.
• Existing methods for quantifying ATPase activity can be time-consuming or require specialized equipment.
• High-affinity calcium-dependent adenosine triphosphatase (Ca2+-ATPase) plays vital roles in calcium homeostasis.

Purpose of the Study:

• To develop and validate a simple, rapid, and cost-effective colorimetric assay for measuring high-affinity Ca2+-ATPase activity.
• To enable spectrophotometric quantification of released phosphate in subcellular fractions.
• To demonstrate the assay's applicability to various biological samples.

Main Methods:

• A one-step colorimetric method using a malachite green/molybdate/polyvinyl alcohol reagent.
• Spectrophotometric quantification of the phosphomolybdate-malachite green complex.
• Validation using isolated rat liver microsomes and adipocyte plasma membranes, comparing results with an isotopic assay.

Main Results:

• The developed colorimetric assay provides a simple and rapid method for measuring Ca2+-ATPase activity.
• Polyvinyl alcohol in the reagent allows direct quantification of released phosphate without protein separation.
• The assay yielded comparable results to isotopic methods for rat liver microsomes and adipocyte plasma membranes.

Conclusions:

• This novel colorimetric assay offers an efficient alternative for measuring high-affinity Ca2+-ATPase activity in subcellular fractions.
• The method's simplicity and speed make it suitable for routine biochemical analysis.
• The assay is potentially applicable to the measurement of other ATPase activities, broadening its utility.