Identification of the catalytic subunit of casein kinase II by affinity labeling with 5'-p-fluorosulfonylbenzoyl adenosine.

Casein kinase II, purified from reticulocytes, was covalently labeled with the ATP affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine. The reaction was monitored by the decrease in enzyme activity and showed saturation kinetics with respect to the sulfonyl compound. This suggested a rapid equilibrium was established between the enzyme and affinity reagent prior to a slower, rate-determining step in the overall inactivation process. The enzyme was protected from the covalent modification by ATP and a series of ATP analogs. Their effectiveness in preventing inactivation by the affinity labeling reagent paralleled their ability to function as inhibitors of the phosphotransferase reaction. When radioactive p-fluorosulfonyl [14C]benzoyl adenosine was used to inactivate the enzyme, the alpha subunit was labeled and incorporation of radioactivity into the alpha subunit was blocked when ADP was included in the reaction mixture. Thus the ATP binding site of casein kinase II was shown to be contained within the domain of the alpha subunit of the alpha 2 beta 2 complex.