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

Alexander A. Stewart, Philip Cohen

    Research output: Contribution to journalArticlepeer-review

    13 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)409-416
    Number of pages8
    JournalMethods in Enzymology
    Volume159
    Issue numberC
    DOIs
    Publication statusPublished - 1988

    Fingerprint

    Dive into the research topics of 'Protein Phosphatase-2B from Rabbit Skeletal Muscle: A Ca<sup>2+</sup>-Dependent, Calmodulin-Stimulated Enzyme'. Together they form a unique fingerprint.

    Cite this