Purification And Partial Characterization Of A Phosphatase On The Thykoid Membrane Of Ipomoea Aquatica

...Nearly all aspects of cell life are regulated by reversible protein phosphorylation. Some examples include metabolic processes, gene regulation, cell cycle control, transport and secretory processes, the organization of the cytoskeleton, and cell adhesion. Chloroplast phosphoprotein were first found in thylakoid membranes by Bennett o The attachment or removal of a phosphate group from a protein may have profound effects on that protein's activities and properties, the reversible phospharylation of the membrane protein in thykaloid is a dymanic equilibrium process. The phospharylation and dephospharylation of protein are controlled by illumination. Nowadays the knowledge about protein phosphatase of thykaloid is not well known. For uncovering the effects of reversible phospharylation on the structure and function of PSII reaction centre, we purified a protein phosphatase associated membrane from the thykaloid membrane of Ipomoea aquatica chloroplasts. In our experiments we studied the enzymology and spectrum characters of the purified phosphatase.In our lab, one kind of protein phosphatase associated thylakoid membrane of pomoea aquatica has been isolated. This enzyme was different with the ones reported in the past. A phosphatase was isolated from the chloroplast thylakoid membrane of Ipomoea aquatica, by NaCl extration, ammonium sulfate precipitation, ion-exchange chromatography and hydrophic chromatography through Butyl-Toyopearl 650M column. The result from SDS-PAGE showed that the enzyme was one protein with molecular weight of 14.9 kD. This phosphatase could catalyzehydrolysis of phosphate monoesters pNPP and had a Km of 1.76 X 10-6mol/L. The optimal pH for the enzyme reaction was 5.0, indicating it was belong to acid phosphatase, while the optimal temperature of enzyme catalysis was 50 C , suggesting it is a thermostable one. EDTA and NaF could inhibit the enzyme activity intensively, however, it was obviously that no effect of NaCl on the phosphatase activity. In order to investigate the effects of pH, temperature, NaF and bivalent cations on the conformation of the phosphatase in solution, we monitored the difference of intrinsic fluorescence of the phosphatase and compared changes of the enzyme activity under those conditions. The tertiary structure loosed and the intensity of fluorescence decreased below pH 6.0. The intensity of fluorescence was lowest at pH 5.0 and the tertiary structure was reconstructed as the solution pH was increased. The characters of fluorescence spectra were similar in the temperature region, 10-30C. The fluorescence intensity of the phosphatase increased, followed by a decreasing process, in the temperature region of 40-90 C. The enzyme was inhibited by NaF significantly and the fluorescence spectra of the protein were influenced obviously by the concentration of NaF. The data from our experiments showed that the bivalent cations can modulate the activity of the phosphatase but maybe is independent of the tertiary structure.