Purification and characterization of the cytoplasmic casein kinase I from rat liver.
Two cyclic nucleotide-independent protein kinases, which preferentially utilize casein and phosvitin as substrates, exist in rat liver. In contrast to cytosol the "light' form of these enzymes was predominant in the "microsomal extract'. This form (30 000-40 000 daltons, casein kinase I) was separated from the "heavy' form (130 000 daltons, casein kinase II) by gel filtration. This enzyme was then purified by successive chromatography on carboxymethyl-Sephadex, phosvitin-Sepharose and hydroxyapatite. The activity of the purified enzyme was 2000-3000-fold the casein kinase activity of the cytosol. It had a s20,w of approx. 3 S as determined on sucrose density gradient. After iodination or incubation with [gamma- 32P]ATP, it was analyzed on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and appeared to be composed of a single polypeptide (36 000 +/- 1000 daltons) which self-phosphorylated. In contrast to casein kinase II, casein kinase I preferentially utilized ATP over GTP. The Km value for ATP was determined to be 14 micro M. The Km value for phosvitin was 0.17 mg/ml. Casein kinase I phosphorylated sites different from those of casein kinase II (as shown with ribosomes or SV40 T antigen). Casein kinase I was further characterized by studying its thermal stability. The half-life at 37 degree C was 6 min and 1 min 30 s at 54 degree C. In the presence of two substrates (ATP and phosvitin), the half-life at 54 degree C increased from 1 min 30 s to 4 min. Hemin strongly increased the rate of inactivation of the casein kinase I at 37 degree C in the absence or presence of the substrates. N-ethylmaleimide also inactivated casein kinase I. Phosvitin, though not ATP, protected the enzyme. This observation may indicate that thiols are involved at the binding site of the enzyme for the protein substrate.
D000818:Animals; D047388:Casein Kinases; D003600:Cytosol / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D006418:Heme / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D006418:Heme; D011494:Protein Kinases; D047388:Casein Kinases; D005033:Ethylmaleimide