Protein Phosphatase Type 1 and Type 2A Assays.

Protein phosphatases type 1 (PP1) and type 2A (PP2A) are the only activities known in mammalian tissues to dephosphorylate glycogen phosphorylase a. Phosphorylase was the first enzyme demonstrated to undergo regulation of catalytic activity via reversible covalent modification involving phosphorylation. It exists in a dephosphorylated, b form, which is catalytically active only in the presence of its allosteric activator, AMP, and a phosphorylated, a form, which is catalytically active in the absence of AMP. The conversion of phosphorylase b to a is catalyzed by phosphorylase kinase, a process that results in the phosphorylation of a single serine residue (Ser-14) in each identical subunit of the native dimer. This is one of the advantages of using phosphorylase a as a protein phosphatase substrate to study PP1 and PP2A activities in mammalian and nonmammalian eucaryotic cells. Other protein substrates, such as glycogen synthase and phosphorylase kinase, contain multiple phosphorylation sites, the reversible covalent modification of which are not always correlated with changes in enzymic activity. Other advantages include the commercial availability of both phosphorylase and phosphorylase kinase Phosphorylase b can also be conveniently isolated in gram quantities from 1 kg of either fresh or frozen rabbit skeletal muscle by a procedure that can be completed within a week and does not involve any chromatography steps (1,2). Phosphorylase kinase can be isolated within a 24 h period from fresh rabbit skeletal muscle (3). [ABSTRACT FROM AUTHOR]