Purification and partial characterization of protein phosphatases from rat thymus

Éva Bakó, V. Dombrádi, F. Erdődi, Lawrence Zumo, Pál Kertai, P. Gergely

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Protein phosphatases assayed with phosphorylase a are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the β subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the α subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycations and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.

Original languageEnglish
Pages (from-to)300-305
Number of pages6
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1013
Issue number3
DOIs
Publication statusPublished - Oct 9 1989

Fingerprint

Phosphoprotein Phosphatases
Thymocytes
Thymus Gland
Protein Phosphatase 1
Phosphoric Monoester Hydrolases
Gel Chromatography
Phosphorylase Kinase
Cytosol
Catalytic Domain
Agarose Chromatography
Protamines
Electrophoresis
Phosphorylase Phosphatase
Hexadimethrine Bromide
Gels
Phosphorylase a
Protein Phosphatase 2
Muscles
Proteins
Liver

Keywords

  • (Rat)
  • Heparin-Sepharose
  • Polycation
  • Protein phosphatase-1
  • Protein phosphatase-2A
  • Thymocyte

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Purification and partial characterization of protein phosphatases from rat thymus. / Bakó, Éva; Dombrádi, V.; Erdődi, F.; Zumo, Lawrence; Kertai, Pál; Gergely, P.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1013, No. 3, 09.10.1989, p. 300-305.

Research output: Contribution to journalArticle

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AU - Gergely, P.

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