title: Purification, subunit composition and regulatory properties of the ATP X Mg2+-dependent form of type I phosphoprotein phosphatase from bovine heart.
abstract: The ATP X Mg2+-dependent phosphoprotein phosphatase has been purified from bovine heart to near-homogeneity. It is a heterodimer (75 kDa) consisting of a catalytic (C) subunit (40 kDa) and a regulatory (R) subunit (35 kDa). The R subunit, which is identical to inhibitor-2, is transiently phosphorylated during activation of the enzyme catalyzed by phosphatase-1 kinase (FA). Maximal activation requires preincubation of the phosphatase with FA and ATP X Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA X ATP X Mg activity, depending on the degree of endogenous proteolytic damage of the phosphatase during purification), but not by either FA or ATP alone. Limited trypsinization results in a rapid and total degradation of the R subunit and partial degradation of the 40-kDa C subunit to active proteins of 35-38 kDa. The resulting 'nicked' C subunit of 35-38 kDa is no longer dependent on FA for activation and can be fully activated by Mg2+ (or Mn2+) alone. Endogenous proteolytic damage of the R subunit also results in an increase of activity that can be expressed by M2+ alone with a concomitant decrease of the FA-dependent activation. Although Mn2+ is slightly more effective than Mg2+ in expressing the holoenzyme basal activity, the activation by Mn2+ is only about 60% of that of Mg2+ when FA and ATP are also present. In the activation by adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), Co2+ is the most effective cofactor. The activation by ATP[gamma S] X Co2+ is more than 50% of that by ATP X Mg2+. The present studies indicate that Mg2+ is the natural divalent cation for the FA-catalyzed activation in which Mg2+ plays two distinctly different roles: it forms Mg2+ X ATP which serves as a substrate for the kinase; it acts as an essential cofactor for the catalytic function of the phosphatase. The discrepancies between the results obtained by this and other laboratories with respect to the effectiveness of Mg2+ and ATP[gamma S] in the activation of the phosphatase are discussed.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology; D054645:Protein Phosphatase 1; D011817:Rabbits; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
chemical_list: D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D008345:Manganese; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C045455:phosphatase-1 kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D008274:Magnesium