Purification and characterization of a protein phosphatase from winged bean

摘要

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92000 and 85000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., and Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone HI (P-hisHl), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., and Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisHl and phosphocasein prepared by using heterologous cAMP- dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisHl. The enzyme is strongly stimulated by a combination of Mg superior 2 superior + and Ca superior 2 superior +, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.