Determination of phosphorylase kinase activity in crude homogenates by affinity chromatography on 5′-AMP Sepharose
✍ Scribed by Niels Borregaard; Viggo Esmann
- Publisher
- Elsevier Science
- Year
- 1980
- Tongue
- English
- Weight
- 320 KB
- Volume
- 105
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
A sensitive method for measuring phosphorylase kinase activity by the incorporation of anP from ]y-"*]ATP into phosphorylase in the presence of other phosphorylation reactions is described. The kinase reaction is carried out in a crude homogenate. After stopping the reaction, a portion of the reaction mixture is withdrawn for assay of phosphorylase conversion and the rest is applied on a 5'-AMP Sepharose column. Phosphorylase in both forms is retained on the column while other phosphorylated proteins and l-y-"*P]ATP are washed out. The phosphorylase is then eluted by 10 mM AMP and the radioactivity incorporated is counted.