The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II.
The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).
D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D006882:Hydroxyapatites; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits
D006882:Hydroxyapatites; D009842:Oligopeptides; D017886:Durapatite; C050151:alanyl-alanyl-alanyl-seryl-phenylalanyl-lysyl-alanyl-lysyl-lysinamide; D011494:Protein Kinases; D006656:Protamine Kinase; D011493:Protein Kinase C