Characterization of a cytosolic phosphatase from pea plumules having significant protein tyrosine phosphatase activity

An enzyme with phosphatase activity has been purified to near homogeneity from the cytosolic fraction of pea (Pisum sativum L. cv Alaska) plumules. The enzyme has an estimated molecular mass of between 42 kDa (SDS-PAGE) and 60 kDa (molecular sieve HPLC). It has an isoelectric point range from 6.2 to 6.5. The enzyme was characterized using para-nitrophenyl phosphate (pNPP) and 32P-labeled proteins as substrates. The enzyme acted as an acid phosphatase (APase, EC 3.1.3.2) with a pH optimum from 4.5 to 6.5 when pNPP was used as substrate. However, its pH optimum shifted to pH 7.0 when 32P-Tyr-poly(Glu,Tyr) was used as substrate, and under these conditions its enzymatic properties were very similar to those of protein tyrosine phosphatases (PTPases, EC 3.1.3.48). The enzyme could be inhibited by micromolar concentrations of molybdate and vanadate, but not by the APase inhibitors citrate and tartrate, or by the protein Ser/Thr phosphatase inhibitors, okadaic acid and microcystin-LR. It did not require Ca2+, Mg2+, or Mn2+ for its activity. In addition to 32P-Tyr-poly(Glu,Tyr), the enzyme could also dephosphorylate 32P-Tyr-myelin basic protein phosphorylated by c-src kinase, but not the P-Tyr-mitogen-activated protein kinase (MAPK) phosphorylated by MAPK kinase. It showed little activity toward 32P-Ser/Thr residues on casein and histone phosphorylated by cAMP-dependent protein kinase (PKA). The sequence of the N-terminal 23 amino acid residues of the phosphatase shows no significant similarity to that of known PTPases from other species, but it had 75 % identity with the N-terminus of a sequence translated from a cDNA of castor bean (Ricinus communis L.). The overall amino acid sequence of the castor seed polypeptide closely matched that of a purple acid phosphatase isolated from kidney bean (Phaseolus vulgaris L.).