Purification, Kinetic and Regulatory Properties of Phosphofructokinase from Chlorella pyrenoidosa

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Phosphofructokinase was purified 585-fold from Chlorella pyrenoidosa by using a combination of ammonium sulphate fractionation, filtration through Sepharose 4B and chromatography on DEAE-Sephacel. Enzyme stability was maintained by the presence of 50 mM P_i at pH 6.6. The optimum pH for activity was 7.7. Concentrations of substrates required to achieve half maximal velocity in the standard assay were 9μM (ATP) and 0.2mM (fructose-6-P). ATP above 0.5mM was inhibitory. Enzyme activity was inhibited by high concentrations (10-100 mM) of P_i, but lower concentrations (1-5 mM) were effective in relieving the influence of other inhibitors such as P-enolpyruvate. Inhibition by P-enolpyruvate was greater at lower pH and with less Pi in reaction mixtures: 50% inhibition could be attained with 0.1 mM P-enolpyruvate. Fructose-2,6-bisphosphate, which was shown to be present in Chlorella, had no effect on the phosphofructokinase. Chlorella appeared to contain only one form of phosphofructokinase, possibly in the chloroplast. No pyrophosphate:D-fructose-6-P 1-phosphotransferase activity could be detected.
日本植物生理学会の論文