Characterization of initial autophosphorylation events in rabbit skeletal muscle phosphorylase kinase.
Initial autophosphorylation of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 8.0 caused an increase in enzymatic activity that closely paralleled phosphorylation of the beta subunit. Peptide maps revealed that the first phosphate incorporated into the beta subunit during autophosphorylation was on the same tryptic peptide previously isolated from phosphorylase kinase that had been phosphorylated by cAMP-dependent protein kinase (Cohen P., Watson, D.C., and Dixon, G.H. (1975) Eur. J. Biochem. 51, 79-92). When preincubated with phosphorylase kinase for one min, Ca2+ and Mg2+ synergistically stimulated subsequent autophosphorylation at pH 6.8. After this treatment phosphorylation of both the alpha and beta subunits became linear, and the first site phosphorylated on the beta subunit at pH 6.8 corresponded to the first site phosphorylated at pH 8.0. Removal of the lag as a consequence of the synergistic action of the metal ions allowed determination of a Km for MgATP of approximately 20 microM during initial autophosphorylation at either pH 6.8 or 8.2. With phosphorylase b as the substrate the Km values for MgATP under identical conditions were determined to be approximately 30 and 60 microM at pH 6.8 and 8.2, respectively. Initial rates of autophosphorylation over a 30-fold range of phosphorylase kinase concentrations suggest that incorporation of the first 1 to 2 mol of phosphate per alpha beta gamma delta tetramer occurs through an intramolecular mechanism.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010748:Phosphopeptides / Q000032:analysis; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D010748:Phosphopeptides; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase; D014357:Trypsin; D008274:Magnesium; D002118:Calcium