COPURIFICATION AND CHARACTERIZATION OF POPPY SEED PHOSPHATASE AND PHOSPHOPROTEIN PHOSPHATASE-ACTIVITIES

A phosphatase (Pase) was purified from poppy seed by a procedure successively involving chromatography on carboxymethyl-Sepharose CL-6B, Ultrogel AcA44 and a SP5PW cation exchanger HPLC column. The Pase has a native molecular weight of 106 000 as determined from gel filtration. Two polypeptides (M(r) 57 000 and 63 000) are observed on SDS-PAGE of the purified Pase. The Pase catalyses the dephosphorylation of p-nitrophenylphosphate (PNP) and of other phosphomonoesters including 3'-AMP, 5'-AMP, 2'-AMP, O-phospho-L-threonine, O-phospho-L-serine and O-phospho-L-tyrosine. ATP and ADP, but not bis-PNP, are substrates for the Pase. Phosphoprotein phosphatase (PrPase) activity determined with serine-phosphorylated phosphopolypeptide substrates, namely phosphohistone III-S, phosphoKKRAARATSNVFA-NH2 and phosphoLRRASLG, exactly copurifies on gel filtration with Pase determined with PNP as substrate. The Pase has a pH optimum of 5.0 with PNP as substrate. The Pase has no absolute requirement for a divalent metal ion activator. The Pase is not inhibited by okadaic acid or microcystin-LR. The Pase is inhibited by millimolar concentrations of phosphate and pyrophosphate and by micromolar concentrations of vanadate and molybdate (IC50 values 4 and 0.1-mu-M, respectively). F-, Zn2+ and Cu2+ inhibit the Pase (IC50 values 300, 60 and 20-mu-M, respectively).