A microassay for ATPase

Richard D. Henkel, John L. VandeBerg, Richard A. Walsh

    Research output: Contribution to journalArticlepeer-review

    110 Scopus citations

    Abstract

    A newly developed microtechnique for quantitating activity of myosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assay (ELISA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELISA plate reader. An entire 96-well microplate can be scanned in less than 2 min, and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 μl, and the ATPase activity of as little as 10 ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique.

    Original languageEnglish (US)
    Pages (from-to)312-318
    Number of pages7
    JournalAnalytical Biochemistry
    Volume169
    Issue number2
    DOIs
    StatePublished - Mar 1988

    Keywords

    • ATPase
    • microassay
    • myosin
    • phosphatase
    • phosphate detection

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Fingerprint Dive into the research topics of 'A microassay for ATPase'. Together they form a unique fingerprint.

    Cite this