Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells.
We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D047388:Casein Kinases; D003600:Cytosol / Q000032:analysis; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000032:analysis*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000596:Amino Acids; D009363:Neoplasm Proteins; D011494:Protein Kinases; D047388:Casein Kinases