Abstract
A phosphatase (Pase) was purified from poppy seed by a procedure succesively 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 bserved 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-NH 2 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 (IC 50 values 4 and 0.1 μM, respectively). F −, Zn 2+ inhibit the Pase (IC 50 values 300, 60 and 20 μM, respectively).
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