Simultaneous measurement of multiple membrane ATPases in microtiter plates

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Erythrocyte membrane-bound ATPases play crucial roles in cellular ion transport and homeostasis.
• Accurate and efficient assays are needed to study these enzymes.
• Existing methods may be time-consuming or require specialized equipment.

Purpose of the Study:

• To describe a detailed, adapted autoanalyzer method for determining erythrocyte membrane-bound ATPases activities.
• To establish a rapid, simple, and nonisotopic assay suitable for microtiter plates.
• To assess the precision and reliability of the developed assay.

Main Methods:

• Adaptation of the Raess and Vincenzi (1980a) autoanalyzer method for microtiter plate format.
• Determination of Mg2+ ATPase, Na+/K+-ATPase, Ca2+ ATPase, and calmodulin-activated Ca2+ ATPase activities.
• Measurement of inorganic phosphate (P(i)) using a modified Fiske and Subbarow method (1925) with a plate reader.

Main Results:

• The assay accurately measures activities of Mg2+ ATPase, Na+/K+-ATPase, Ca2+ ATPase, and calmodulin-activated Ca2+ ATPase.
• High intra-assay precision was demonstrated for all measured ATPases (CVs ranging from 3.2% to 5.7%).
• The assay is performed entirely within a single microtiter plate, minimizing handling and errors.

Conclusions:

• The developed microtiter plate assay is a rapid, simple, and nonisotopic method for quantifying erythrocyte membrane-bound ATPases.
• This assay offers improved efficiency and reduced error compared to traditional methods.
• It provides a reliable tool for biochemical and cell biology research involving membrane-bound ATPases.