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

The Arabidopsis thaliana type I protein phosphatase (PPI) 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 PPI 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 I protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC50 = 0.65 nM), tautomycin (IC50 = 0.06 nM), microcystin-LR IC50 = 0.01 nM), nodularin (IC50 = 0.035 nM), calyculin A (IC50 = 0.09 nM), okadaic acid (IC50 = 20 nM) and cantharidin (IC50 = 60 nM). The enzyme was also inhibited by fostriecin (IC50 = 22 muM), NaF (IC50 = 2.1 MW Pi (IC50 = 9.5 mM), and PPi (IC50 = 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 PPI targeting subunits. (C) 2001 Elsevier Science B.V. All rights reserved.