Activation of bovine heart ATP-MG2+-dependent phosphoprotein phosphatase: isolation of a phosphoenzyme intermediate and its conversion to the active form via a Mg2+-dependent autodephosphorylation reaction

• Published in: Biochemical and biophysical research communications Vol. 128; no. 3; pp. 1203 - 1210
• Main Authors: PRICE, D. J, HENG-CHUN LI
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier 16.05.1985
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Applied sciences; Cattle; Enzyme Activation - drug effects; Exact sciences and technology; In Vitro Techniques; Magnesium - pharmacology; Myocardium - enzymology; Other techniques and industries; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Phosphatase 1
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(85)91068-X

The ATP-Mg2+-dependent protein phosphatase, a holoenzyme form of type I protein phosphatase (phosphatase-1) requires the action of phosphatase-1 kinase (FA) for activation. The enzyme (75 kDa) purified from bovine heart consists of a catalytic (C) and a regulatory (R) subunit of 40 kDa and 34 kDa, respectively, and activation is associated with phosphorylation of the R-sub-unit. A procedure has been developed for isolation of [32P]phosphatase-1 ( [32P]E-P) in non-denatured form. In the absence of divalent cation, [32P]E-P is catalytically inactive and the phosphorylation is stable. Addition of Mg2+ triggers autodephosphorylation of [32P]E-P with concomitant generation of phosphorylase phosphatase activity. The autodephosphorylation/activation process is dependent on Mg2+ concentration. The KA value for Mg2+ is 0.6 mM. The phosphorylase phosphatase activity generated from the release of 1 mol. 32P is 1.1 X 10(12) units which is equivalent to 15,000 units per mg enzyme protein. The present findings provide direct evidence that the phosphorylated phosphatase-1 is not the active form (Ea). Instead, Ea is directly produced from the intermediate by a Mg2+-dependent autodephosphorylation reaction.