Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments.
Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.
D000818:Animals; D047388:Casein Kinases; D002417:Cattle; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010206:Papain; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D011494:Protein Kinases; D047388:Casein Kinases; D010206:Papain