Isolation and sequence of a tryptic peptide containing the autophosphorylation site of the regulatory subunit of bovine brain protein kinase II.
The regulatory subunit (RII-B) of bovine brain protein kinase II and the well-characterized regulatory subunit of heart protein kinase II (RII-H) exhibit similar physicochemical properties, but differ significantly in their peptide maps and antigenic determinants. As a starting point for studying structure/function relationships in RII-B and investigating the extent of homology and diversity between RII-B and RII-H, a peptide containing the autophosphorylation site of RII-B has been characterized. The phosphopeptide was rapidly (36 h) purified to homogeneity (yield = 40%) from a tryptic digest of RII-B using three consecutive reverse-phase high performance liquid chromatography steps. A combination of gas-phase microsequencing and solid-phase Edman degradation was used to determine the sequence and to identify the phosphorylated site: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-A la-Glu. RII-B contains a classical phosphorylation site for the catalytic subunit, and the phosphopeptide sequence is homologous to the sequence surrounding the phosphorylation site of RII-H. Fourteen amino acids are identical in the two sequences, and the high net negative charge on the peptide is conserved. However, the peptide from RII-B is alanine-rich and more hydrophobic. Furthermore, five differences between the two functionally related sequences provide direct evidence for the idea that RII-B and RII-H are the products of related but distinct genes.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification*; D010748:Phosphopeptides / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases