Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides.
The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.
Litchfield
D W
DW
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Lozeman
F J
FJ
Piening
C
C
Sommercorn
J
J
Takio
K
K
Walsh
K A
KA
Krebs
E G
EG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D047388:Casein Kinases; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006882:Hydroxyapatites; D015151:Immunoblotting; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010449:Peptide Mapping; D011494:Protein Kinases / Q000302:isolation & purification*; D012689:Sequence Homology, Nucleic Acid; D013737:Testis / Q000201:enzymology*
D000906:Antibodies; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D009842:Oligopeptides; D017886:Durapatite; D011494:Protein Kinases; D047388:Casein Kinases