Catalytic and molecular properties of a highly purified G type casein kinase from bovine lung tissue

A preparation procedure has been worked out to obtain a highly purified G type (using GTP as well as ATP) casein kinase from large quantities of bovine lung tissue. It included ion-exchange (DEAE and phosphocellulose) and affinity (casein and ATP-Sepharose) chromatography combined with a flocculation step, and yielded an apparently homogeneous preparation with a 16% yield and a purification factor of more than 1400. The purified lung casein kinase used GTP (Km 16 microM) almost as well as ATP (Km 6.7 microM) and exhibited the major catalytic properties of the casein kinase G previously described in bovine adrenal cortex (Cochet, C., Job, D., Pirollet, F. and Chambaz, E.M. (1981) Biochim. Biophys. Acta 658, 191-201). Mg2+ (30-50 mM) and spermine (2 mM) were potent activators of lung casein kinase G activity, whereas the enzyme was inhibited by heparin and quercetin. The purified enzyme underwent self-phosphorylation in the presence of ATP or GTP, serine being the only target amino acid under these conditions, whereas both serine and threonine were phosphorylated by the enzyme in casein. Lung casein kinase G exhibited an apparent molecular weight between 140 000-160 000 upon gel filtration and appeared formed by the association of two different subunits upon SDS-polyacrylamide gel electrophoresis. The two subunits of Mr 38 000 (alpha) and 27 000 (beta) exhibited a 2:1 ratio upon quantitative scanning, suggesting an alpha 3 beta 2 combination in the oligomeric native enzyme structure. Peptide mapping of the two isolated subunits following 125I-labeling and papain digestion did not disclose any common fragment. The casein kinase catalytic activity was found associated with the alpha (38 kDa) enzyme subunit after recovery from gel electrophoresis in the presence of SDS, whereas the 27 kDa (beta) subunit was the major target of the enzyme self-phosphorylation reaction. alpha and beta subunits appeared strongly associated in the oligomeric enzyme and the possible role of the beta subunit in the casein kinase G activity remains to be examined. The purified casein kinase G, which can be obtained by the present procedure, should facilitate the study of the biological significance of this phosphorylation system in the intact cell.