Nonactivated phosphorylase kinase is a phosphoprotein: differentiation of two classes of endogenous phosphoserine residues by phosphorus-31 nuclear magnetic resonance spectroscopy and phosphatase sensitivity.
A standard preparation of phosphorylase kinase from rabbit skeletal muscle contains 2 mol of phosphoserine/mol of alpha beta gamma delta. This basal stoichiometry is not influenced by application of propranolol and insulin in vivo; these serine phosphates could not be hydrolyzed by phosphatases of the muscle extract or by alkaline phosphatases. When the enzyme is purified in the presence of the protein phosphatase inhibitor sodium fluoride, it contains either 1 or 3 additional mol of phosphoserine/mol of alpha beta gamma delta, termed phosphatase-sensitive phosphates. Both classes of phosphates yield in formic acid one single 31P NMR signal of a narrow line width (approximately 3 Hz) very similar in chemical shift to free phosphoserine. Phosphoserine is also identified by its chemical shift when dissolved in 8 M guanidinium chloride and by its electrophoretic mobility after acid hydrolysis. By self-phosphorylation of phosphorylase kinase, 14 additional mol of phosphate/mol of alpha beta gamma delta was incorporated, and all were identified as phosphoserine by 31P NMR spectroscopy. In native phosphorylase kinase, the 31P NMR signals of both the basal and the phosphatase-sensitive phosphates are substantially broadened and reduced in intensity, indicating strong interactions of the phosphate groups with the protein. The basal and phosphatase-sensitive phosphates give in 8 M guanidinium chloride a homogeneous NMR signal above pH 6; it splits into a doublet below pH 6 and into a triplet below pH 5.(ABSTRACT TRUNCATED AT 250 WORDS)
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010768:Phosphoserine / Q000032:analysis*; D012694:Serine / Q000031:analogs & derivatives*; D012969:Sodium Fluoride / Q000494:pharmacology
D010750:Phosphoproteins; D010768:Phosphoserine; D012694:Serine; D012969:Sodium Fluoride; D010764:Phosphorylase Kinase; D000469:Alkaline Phosphatase