Protein Phosphatase-2B from Rabbit Skeletal Muscle: A Ca²⁺-Dependent, Calmodulin-Stimulated Enzyme

Protein phosphatase-2B (PP-2B), a Ca²⁺-dependent enzyme that is stimulated by calmodulin, can be purified from rabbit skeletal muscle. This chapter discusses the procedure for this purification. PP-2B is most conveniently assayed by its ability to dephosphorylate inhibitor-l, although other substrates, such as phosphorylase kinase, may also be used. PP-2B accounts for 75–80% of the inhibitor-I phosphatase activity in skeletal muscle extracts when assays are performed at 3μM Ca²⁺. The enzyme does not precipitate with the glycogen-protein particles when muscle extracts are adjusted to pH 6.1 and is therefore recovered in the pH 6.1 supernatant. At this stage, activity is not dependent on calmodulin, but is activated 4-fold by this protein after fractionation with ammonium sulfate, and 10-fold after the first chromatography on DEAE Sepharose. The final stage of purification involves adsorption to calmodulin- Sepharose in the presence of Ca²⁺, followed by elution with EGTA. However, incubation of the enzyme with Ca²⁺ at this stage causes a rapid irreversible loss of activity unless a carrier protein such as phosphorylase b or serum albumin is added.