A rapid and sensitive assay method for protein kinase

✍ Scribed by Kuo-Ping Huang; J.C. Robinson

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 421 KB
• Volume: 72
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(76)90571-6

✦ Synopsis

A simplified assay method is described for the determination of protein kinase activity. Enzymatic activity is followed by measuring the incorporation of 32P from the terminal phosphoryl group of nucleoside triphosphates into protein substrate. Separation of the resulting 32P-labeled phosphoprotein from the unreacted 3ZP-labeled nucleoside triphosphate is achieved by chromatography of the reaction mixture on Gelman thin-layer chromatography medium for 5 min. The assay is shown to be more accurate than previous methods. Some properties of cyclic AMP-dependent and cyclic AMP-independent protein kinases from rabbit muscle are compared by using the assay method. METHODS Calf thymus histone, type II-A (histone mixture), type III-S (lysinerich fraction), and type VIII-S (arginine-rich fraction); protamine; phosvitin; ATP; GTP; trypsin; and subtilisin were purchased from Sigma Chemical Company. [y-32P]ATP and [-Y-~~P]GTP were obtained from 593