Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1)

Biochim Biophys Acta. 2001 Nov 26;1550(1):52-63. doi: 10.1016/s0167-4838(01)00267-9.


The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / chemistry
  • Arabidopsis / enzymology*
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / chemistry
  • Phosphoprotein Phosphatases / isolation & purification*
  • Plant Extracts / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Enzyme Inhibitors
  • Peptide Fragments
  • Plant Extracts
  • Phosphoprotein Phosphatases