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Related Experiment Videos

A microassay for ATPase.

R D Henkel1, J L VandeBerg, R A Walsh

  • 1Southwest Foundation for Biomedical Research, San Antonio, Texas 78284-9950.

Analytical Biochemistry
|March 1, 1988
PubMed
Summary
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A new microtechnique offers a sensitive and rapid method for quantifying myosin ATPase activity. This assay, suitable for 96-well plates, enables detailed analysis of cardiac myosin from small tissue samples.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Myosin ATPase activity is crucial for muscle contraction.
  • Existing spectrophotometric methods for ATPase quantification are time-consuming and less sensitive.
  • Analysis of cardiac myosin often requires large tissue samples or less sensitive assays.

Purpose of the Study:

  • To develop and validate a novel microtechnique for quantifying myosin ATPase activity.
  • To enhance sensitivity and reduce assay time compared to existing methods.
  • To enable detailed kinetic analysis of cardiac myosin from small tissue specimens.

Main Methods:

  • A microassay was developed for measuring myosin ATPase (EC 3.6.1.32) activity in 96-well microplates.
  • The assay involves incubating purified myosin with ATP, followed by termination and measurement of inorganic phosphate using malachite green.

Related Experiment Videos

  • Quantification is performed using a microELISA plate reader, with data transferable for analysis.

    • Main Results:

    • The microassay is highly sensitive, detecting 0.2–3 nmol of inorganic phosphate in 50 µl.
    • It can measure ATPase activity from as little as 10 ng of rat cardiac myosin.
    • Assay completion and microplate scanning take less than 2 minutes per plate.

    Conclusions:

    • The developed microtechnique provides a sensitive, rapid, and efficient method for myosin ATPase activity quantification.
    • This microassay facilitates detailed enzymatic and kinetic analysis of cardiac myosin using minimal tissue samples.
    • The technique is advantageous for large-scale studies involving numerous small specimens.