ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor.
An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012265:Ribonucleotides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011506:Proteins; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D008274:Magnesium