An improved purification procedure and properties of casein kinase II from brain.
A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that the Km for ATP is 12.5 microM and 25.1 microM when using phosvitin and casein respectively as phosphoryl acceptors. The Km for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while the Vmax is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 microgram/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.
Alcázar
A
A
Dpto Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Martin
E
E
López-Fando
J
J
Salinas
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology*; D047388:Casein Kinases; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D011494:Protein Kinases / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000032:analysis
D002412:Cations; D004791:Enzyme Inhibitors; D006493:Heparin; D011494:Protein Kinases; D047388:Casein Kinases