Stimulation of phosphorylase phosphatase activity of protein phosphatase 2A(1) by protamine is ionic strength dependent and involves interaction of protamine with both substrate and enzyme

The effects of protamine on the phosphorylase phosphatase activity of porcine cardiac protein phosphatase 2A(1) (PP2A(1)) were complex and ionic strength dependent. Under ionic strength conditions that protamine activation was optimal, activation of PP2A(1) by either dilution or heparin was prevented. A time-dependent deactivation of the protamine-stimulated phosphatase activity was observed when PP2A(1) was preincubated with protamine. Protamine forms a very tight association with phosphorylase a, which is optimal at a 1:1 protamine:phosphorylase a monomer molar ratio. Protamine activation of PP2A(1) activity, however, is not substrate-directed since the basic polypeptide did not stimulate either the activity of the catalytic subunit or trypsinolysis of [P-32]phosphorylase a. The interaction of protamine with phosphorylase a does not apparently involve the phosphorylation site in the protein substrate (ser 14). The activation of PP2A(1) by protamine is proposed to involve part of the basic polypeptide, not associated with phosphorylase a monomer, interacting with the regulatory and/or the catalytic subunit(s) of the phosphatase. A minimal model for the activation of PP2A(1) by protamine was tested kinetically. In this model, free PP2A(1) binds with decreasing affinities to the protamine:phosphorylase a complex, free phosphorylase a, and free protamine. Protamine decreases the K-m of PP2A(1) for the phosphorylase a monomer 5-fold and increases the V-max 17-fold. Interaction of free protamine with PP2A(1) inhibits the phosphatase activity.