Identification of the catalytic subunit of an oligomeric casein kinase (G type). Affinity labeling of the nucleotide site using 5'-[p-(fluorosulfonyl)benzoyl]adenosine.
Identification of the catalytic subunit of a G type [using guanosine 5'-triphosphate (GTP) as well as adenosine 5'-triphosphate (ATP) as phosphate donor], oligomeric, cyclic nucleotide independent casein kinase purified from bovine lung was carried out after reaction with 5'-[p-(fluorosulfonyl)-benzoyl]adenosine (FSBA) and isolation of the subunit components of the enzyme. FSBA exhibited the major characteristics of an affinity label reacting at the nucleotide (ATP, GTP) site of the casein kinase. FSBA acted as a competitive inhibitor of ATP (and GTP), led to complete inactivation of the enzyme in a reaction showing two kinetic steps, and became irreversibly bound to the protein. After being labeled with FSBA, the casein kinase (apparent molecular weight of 140 000) was separated into its two monomeric components of apparent molecular weights 38 000 (alpha) and 27 000 (beta), respectively, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of radioactive FSBA showed that specific affinity labeling was limited to the alpha casein kinase subunit. This result was in agreement with the fact that casein kinase activity was found associated with the alpha monomer after electrophoretic separation of the alpha and beta subunits. It may thus be concluded that the largest (alpha) subunit contains the catalytic site of the casein kinase G. Electrophoretic analysis of purified protein kinase under denaturing conditions suggested an alpha 3 beta 2 combination for an apparent molecular weight of 130 000-140 000. However, a maximum of 2 mol of FSBA could be specifically bound to the alpha subunit per mol of enzyme, with a concomitant complete inactivation. These data would be in agreement with an alpha 2 beta 2 subunit composition for casein kinase G, as proposed by other research groups for a similar type of protein kinase of different sources. These observations suggest that the alpha subunits are functionally similar, each of them containing a nucleotide (ATP, GTP) binding site. The possible role of the beta subunit in the enzyme activity remains to be established.
D000241:Adenosine / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D047388:Casein Kinases; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism
D000345:Affinity Labels; D009711:Nucleotides; D047428:Protein Kinase Inhibitors; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D011494:Protein Kinases; D047388:Casein Kinases; D000241:Adenosine