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Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.

Publication ,  Journal Article
Campos, M; Fadden, P; Alms, G; Qian, Z; Haystead, TA
Published in: J Biol Chem
November 8, 1996

Biotinylated microcystin was used to affinity purify over avidin-Sepharose the entire cellular content of active forms of protein phosphatase (PP) 1 and 2A holoenzymes present in three subcellular fractions of skeletal muscle. Biotinylated microcystin displayed IC50 values in the nM range against PP-1C (1.58 +/- 0.6 nM S.E., n = 3), PP-2AC (0.63 +/- 0.2 nM S.E., n = 3) and SMPP-1M (5.9 +/- 1.3 S.E., n = 3). Subsequent anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis of the microcystin-biotin eluates of the three fractions revealed a complex pattern of proteins associated with PP-1C and PP-2AC. Far Western analysis and the rebinding interaction with recombinant PP-1C distinguished proteins in the eluates that bound PP-1C from those that bound PP-2AC. In Far Western analysis, 29 distinct proteins were identified to bind PP-1C. Significantly, these same proteins, plus seven others, were also recovered from the isothiocyanate eluates from microcystin-Sepharose by a rebinding interaction with PP-1C-microcystin-biotin. The number of proteins and range of novel molecular masses (18-125 kDa) identified to interact with PP-1C by these two techniques cannot be accounted for by the previously characterized subunits of PP-1. Our findings further support the concept that PP-1C is regulated in vivo by multiple and distinct substrate-targeting subunits.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

November 8, 1996

Volume

271

Issue

45

Start / End Page

28478 / 28484

Location

United States

Related Subject Headings

  • Rats, Wistar
  • Rats
  • Protein Phosphatase 1
  • Phosphoprotein Phosphatases
  • Peptides, Cyclic
  • Muscle, Skeletal
  • Microcystins
  • Male
  • Kinetics
  • Electrophoresis, Polyacrylamide Gel
 

Citation

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Campos, M., Fadden, P., Alms, G., Qian, Z., & Haystead, T. A. (1996). Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography. J Biol Chem, 271(45), 28478–28484. https://doi.org/10.1074/jbc.271.45.28478
Campos, M., P. Fadden, G. Alms, Z. Qian, and T. A. Haystead. “Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.J Biol Chem 271, no. 45 (November 8, 1996): 28478–84. https://doi.org/10.1074/jbc.271.45.28478.
Campos M, Fadden P, Alms G, Qian Z, Haystead TA. Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography. J Biol Chem. 1996 Nov 8;271(45):28478–84.
Campos, M., et al. “Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.J Biol Chem, vol. 271, no. 45, Nov. 1996, pp. 28478–84. Pubmed, doi:10.1074/jbc.271.45.28478.
Campos M, Fadden P, Alms G, Qian Z, Haystead TA. Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography. J Biol Chem. 1996 Nov 8;271(45):28478–28484.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

November 8, 1996

Volume

271

Issue

45

Start / End Page

28478 / 28484

Location

United States

Related Subject Headings

  • Rats, Wistar
  • Rats
  • Protein Phosphatase 1
  • Phosphoprotein Phosphatases
  • Peptides, Cyclic
  • Muscle, Skeletal
  • Microcystins
  • Male
  • Kinetics
  • Electrophoresis, Polyacrylamide Gel