IDENTIFICATION OF 2 THYLAKOID-ASSOCIATED PHOSPHATASES WITH PROTEIN PHOSPHATASE-ACTIVITY IN CHLOROPLASTS OF THE SOYBEAN (GLYCINE-MAX)

Two thylakoid-associated phosphatases from soybean (Glycine max) have been purified and characterized. Both enzymes were prepared by ultraso nication of thylakoids, (NH4)(2)SO4 fractionation, gel filtration, cat ion, and anion exchange chromatography. Phosphatase I and II copurifie d during the first steps of purification and were separated by ion exc hange chromatography on CM cellulose. The phosphatases have a molecula r mass of 70 kDa. They exhibit a pH optimum at 5.5 and 6, respectively . Mg2+ ions are not required for activity. Several phosphorylated subs trates are hydrolyzed, including phenyl and 4-nitrophenyl phosphate, 3 -phosphoglycerate, and fructose 1,6-bisphosphate. The thylakoid-associ ated phosphatases also possess phosphoprotein phosphatase activity tow ards P-32-labeled casein and chloroplast LHC II as substrates. Fluorid e inhibited the hydrolysis of nitrophenylphosphate and [P-32] casein b ut not the protein phosphatase activity towards P-32-LHC II. The prote in serine/threonine phosphatase inhibitor okadaic acid did not affect these phosphatases at all.