Synthesis and Use of the Protein Phosphatase Affinity Matrices Microcystin-Sepharose and Microcystin-Biotin-Sepharose

  • Greg B. G. Moorhead
  • Timothy A. J. Haystead
  • Carol MacKintosh
Part of the Methods in Molecular Biology book series (MIMB, volume 365)


Microcystin-based affinity matrices have been utilized to demonstrate the association of signaling proteins with protein phosphatases and for the purification of low-abundance microcystin-sensitive protein phosphatases. Here, we describe the procedure for the synthesis and use of microcystin-Sepharose and microcystin-biotin-Sepharose.

Key Words

Microcystin okadaic acid protein phosphatase biotin affinity chromatography cyclic-peptide hepatotoxin cyanobacteria 


  1. 1.
    Cohen, P. T. (2002) Protein phosphatase 1: targeted in many directions. J. Cell. Sci. 115, 241–256.PubMedGoogle Scholar
  2. 2.
    Bollen, M. (2001) Combinatorial control of protein phosphatase-1. Trends Biochem. Sci. 26, 426–431.PubMedCrossRefGoogle Scholar
  3. 3.
    Janssens, V. and Goris, J. (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439.PubMedCrossRefGoogle Scholar
  4. 4.
    Cohen, P. T. (1997) Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem. Sci. 22, 245–251.PubMedCrossRefGoogle Scholar
  5. 5.
    Shenolikar, S. (1994) Protein serine/threonine phosphatases—new avenues for cell regulation. Annu. Rev. Cell Biol. 10, 55–86.PubMedCrossRefGoogle Scholar
  6. 6.
    MacKintosh, C. and MacKintosh, R. W. (1994) Inhibitors of protein kinases and phosphatases. Trends Biochem Sci. 19, 444–448.PubMedCrossRefGoogle Scholar
  7. 7.
    MacKintosh, R. W., Dalby, K. N., Campbell, D. G., Cohen, P. T., Cohen, P., and MacKintosh, C. (1995) The cyanobacterial toxin toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1. FEBS Lett. 371, 236–240.PubMedCrossRefGoogle Scholar
  8. 8.
    Moorhead, G. B. G., MacKintosh, R. W., Morrice, N., Gallagher, T., and Mackintosh, C. (1994) Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography. FEBS Lett. 356, 46–50.PubMedCrossRefGoogle Scholar
  9. 9.
    Campos, M., Fadden, P., Alms, G., Qian, Z., and Haystead, T. A. (1996) Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography. J Biol Chem. 271, 28,478–28,484.PubMedCrossRefGoogle Scholar
  10. 10.
    Moorhead, G., Johnson, D., Morrice, N., and Cohen, P. (1998) The major myosin phosphatase in skeletal muscle is a complex between the β-isoform of protein phosphatase-1 and the MYPT2 gene product. FEBS Lett. 438, 141–144.PubMedCrossRefGoogle Scholar
  11. 11.
    Moorhead, G. B. G., MacKintosh, C., Morrice, N., and Cohen, P. (1995) Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography. FEBS Lett. 362, 101–105.PubMedCrossRefGoogle Scholar
  12. 12.
    Johnson, D. F., Moorhead, G. B. G., Caudwell, F. B., et al. (1996) Identification of protein phosphatase-1-binding domains on the glycogen and myofibrillar targetting subunits. Eur J Biochem 239, 317–325.PubMedCrossRefGoogle Scholar
  13. 13.
    Boudrez, A., Evens, K., Beullens, M., Waelkens, E., Stalmans, W., and Bollen, M. (1999) Identification of MYPT1 and NIPP1 as subunits of protein phosphatase 1 in rat liver cytosol. FEBS Lett. 455, 175–178.PubMedCrossRefGoogle Scholar
  14. 14.
    Meek, S., Morrice, N., and MacKintosh, C. (1999) Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A. FEBS Lett. 457, 494–498.PubMedCrossRefGoogle Scholar
  15. 15.
    Stubbs, M. D., Tran, H., Atwell, A. J., Smith, C. S., Olson, D., and Moorhead, G. B. G. (2001) Purification and properties of Arabidopsis thaliana type one protein phosphatase (PP1). Biochim. Biophys. Acta 1550, 52–63.PubMedCrossRefGoogle Scholar
  16. 16.
    Tran, H. T., Ulke, A., Morrice, N., Johannes, C. J., and Moorhead, G. B. (2004) Proteomic characterization of protein phosphatase complexes of the mammalian nucleus. Mol. Cell. Proteomics 3, 257–265.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Greg B. G. Moorhead
    • 1
  • Timothy A. J. Haystead
    • 2
  • Carol MacKintosh
    • 3
  1. 1.Department of Biological SciencesUniversity of CalgaryCalgaryCanada
  2. 2.Department of Pharmacology and Cancer BiologyDuke University Medical CenterDurhamUSA
  3. 3.MRC Protein Phosphorylation Unit, School of Life SciencesUniversity of DundeeDundeeScotland

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