Purification, subunit composition and regulatory properties of the ATP · Mg2+‐dependent form of type I phosphoprotein phosphatase from bovine heart

Publisher Website
Google Scholar
Abstract
The ATP · Mg2+‐dependent phosphoprotein phosphatase has been purified from bovine heart to nearhomogeneity. 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 · Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA· ATP · Mg activity, depending on the degree of endogeneous 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′‐[γ‐thio]triphosphate (ATP[γS]), Co2+ is the most effective cofactor. The activation by ATP[γS] · Co2+ is more than 50% of that by ATP · 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: (a) it forms Mg2+· ATP which serves as a substrate for the kinase; (b) 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[γS] in the activation of the phosphatase are discussed.

This publication has 36 references indexed in Scilit: