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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
A continuous spectrophotometric assay for phosphorylase kinase
Z X Wang1, Q Cheng, S D Killilea
1National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Analytical Biochemistry
|September 1, 1995
Summary
A new continuous spectrophotometric assay was developed to measure phosphorylase kinase activity. This assay provides kinetic parameters like Km and kcat for nonactivated phosphorylase kinase.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Phosphorylase kinase is a key enzyme in glycogen metabolism.
- Accurate kinetic analysis requires robust assay methods.
Purpose of the Study:
- To develop and validate a continuous spectrophotometric assay for phosphorylase kinase.
- To determine kinetic parameters (Km, kcat) for nonactivated phosphorylase kinase at pH 7.0.
Main Methods:
- A coupled enzyme system using protein phosphatase and purine nucleoside phosphorylase was employed.
- Spectrophotometry was used to quantify inorganic phosphate production.
- The assay was standardized and validated for reliability.
Main Results:
- The assay successfully determined the initial reaction rates of phosphorylase kinase.
- Kinetic parameters were determined: Km = 15.36 ± 0.2 μM (phosphorylase b monomer) and kcat = 21 ± 1.12 s⁻¹.
- The effects of reaction components on enzyme activities were investigated.
Conclusions:
- A continuous, coupled spectrophotometric assay is effective for studying phosphorylase kinase kinetics.
- The developed assay allows for precise kinetic analysis of nonactivated phosphorylase kinase.
- This method provides valuable insights into enzyme regulation and function.
