Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform

B. A. Perrino, Y. L. Fong, D. A. Brickey, Y. Saitoh, Y. Ushio, K. Fukunaga, E. Miyamoto, Thomas Soderling

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    Abstract

    Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The K(m) of calcineurin A for 32P-R(II) pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 μM with or without calmodulin, and calmodulin increased the V(max) about 4-fold. The K(m) of reconstituted calcineurin A plus B for 32P-R(II) pep was 20 μM, and calmodulin increased the V(max) 18-fold without affecting the K(m). CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 μM and 90 μM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.

    Original languageEnglish (US)
    Pages (from-to)15965-15969
    Number of pages5
    JournalJournal of Biological Chemistry
    Volume267
    Issue number22
    Publication statusPublished - 1992

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    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Perrino, B. A., Fong, Y. L., Brickey, D. A., Saitoh, Y., Ushio, Y., Fukunaga, K., ... Soderling, T. (1992). Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. Journal of Biological Chemistry, 267(22), 15965-15969.