CHARACTERIZATION OF THE PHOSPHATASE FROM SPINACH THYLAKOIDS

The protein kinase activity of spinach thylakoid membrane has been identified and widely studied. However, its reverse reaction, catalyzed by the protein phosphatase, still requires more detailed exploration. In this communication, we have investigated the biochemical characterization of the protein phosphatase of thylakoids using different exogenous substrates, including phosphoserine, phosphothreonine and p-nitrophenol phosphate. The thylakoid phosphatase activity was light-independent but NaF sensitive. The optimal pH values of 6.5, 6.5 and 8.0, were observed for these three exogenous substrates, respectively. The thylakoid phosphatase was slightly stimulated by low concentrations of Mg2+, Mn2+ and Ca2+, but inhibited to different degrees at higher concentrations. Tartaric acid and vanadate were found to be inhibitory to the enzymatic activity of thylakoid phosphatase. Sulfhydryl-directed reagent, N-phenylmaleimide, only inhibited 20% of the phosphatase activity, while N-ethylmaleimide exerted no inhibitory effect. The phosphatase is more sensitive to dicyclohexylcarbodiimide than its hydrophilic derivative 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The K(m) and V(max) values of the thylakoid phosphatase under different conditions were determined. The functional size of 54 approximately 61 kD was obtained by radiation inactivation.