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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
A dithio-coupled kinase and ATPase assay
Taurai Chiku1, Phani Kumar Pullela, Daniel S Sem
1Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201-1881, USA.
Journal of Biomolecular Screening
|September 1, 2006
Summary
A novel continuous assay detects adenosine diphosphate (ADP) using ATPbetaS and dithio reagents, enabling universal enzyme activity screening. This method is suitable for high-throughput screening of kinases and ATPases.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Assay development
Background:
- Kinases and ATPases generate adenosine diphosphate (ADP) as a common product.
- A universal assay for ADP detection could facilitate enzyme activity screening.
- Adenosine triphosphate beta-S (ATPbetaS) can serve as a substrate for many kinases.
Purpose of the Study:
- To develop a continuous assay for detecting ADP using ATPbetaS as a substrate.
- To enable activity-based screening of enzyme families that produce ADP.
- To validate the assay for kinetic analyses and high-throughput screening.
Main Methods:
- Utilized ATPbetaS as a substrate and dithio reagents for ADPbetaS detection.
- Employed Ellman's reagent (DTNB) and a fluorescent dithio reagent (DSSA) for thiol detection.
- Performed steady-state kinetic analyses and assessed assay suitability for high-throughput screening (Z' value).
Main Results:
- Developed a continuous assay detectable by absorbance and fluorescence.
- Successfully performed kinetic analyses of hexokinase and myosin ATPase.
- Demonstrated assay's ability to show diastereoselectivity for ATPbetaS isomers.
- Achieved an average Z' value of 0.54 in fluorescence mode, indicating high-throughput screening suitability.
Conclusions:
- The developed assay provides a universal method for screening enzyme activity.
- The assay is adaptable for both absorbance and fluorescence detection.
- The assay is robust and suitable for high-throughput screening applications in enzyme research.
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