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

    Research output: Contribution to journalArticle

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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
    StatePublished - 1992

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    Calcineurin
    Baculoviridae
    Calmodulin
    Phosphoric Monoester Hydrolases
    Protein Isoforms
    Affinity chromatography
    Sf9 Cells
    Agarose Chromatography
    Peptides
    Affinity Chromatography
    Edetic Acid
    Sepharose
    Inhibitory Concentration 50
    Rats
    Brain
    Complementary DNA
    Substrates
    Enzymes

    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.

    Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. / Perrino, B. A.; Fong, Y. L.; Brickey, D. A.; Saitoh, Y.; Ushio, Y.; Fukunaga, K.; Miyamoto, E.; Soderling, Thomas.

    In: Journal of Biological Chemistry, Vol. 267, No. 22, 1992, p. 15965-15969.

    Research output: Contribution to journalArticle

    Perrino, BA, Fong, YL, Brickey, DA, Saitoh, Y, Ushio, Y, Fukunaga, K, Miyamoto, E & Soderling, T 1992, 'Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform', Journal of Biological Chemistry, vol. 267, no. 22, pp. 15965-15969.
    Perrino BA, Fong YL, Brickey DA, Saitoh Y, Ushio Y, Fukunaga K et al. Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. Journal of Biological Chemistry. 1992;267(22):15965-15969.
    Perrino, B. A. ; Fong, Y. L. ; Brickey, D. A. ; Saitoh, Y. ; Ushio, Y. ; Fukunaga, K. ; Miyamoto, E. ; Soderling, Thomas. / Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 22. pp. 15965-15969.
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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.",
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    T1 - Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform

    AU - Perrino, B. A.

    AU - Fong, Y. L.

    AU - Brickey, D. A.

    AU - Saitoh, Y.

    AU - Ushio, Y.

    AU - Fukunaga, K.

    AU - Miyamoto, E.

    AU - Soderling, Thomas

    PY - 1992

    Y1 - 1992

    N2 - 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.

    AB - 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.

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