Phosphofructokinase was purified 585-fold fromChlorella pyrenoidosaby 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 Piat 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 0.2 mM (fructose-6-P). ATP above 0.5 mM was inhibitory. Enzyme activity was inhibited by high concentrations (10–100 mM) of Pibut 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 Piin reaction mixtures: 50inhibition could be attained with 0.1 mmP-enolpyruvate. Fructose-2,6-bisphosphate, which was shown to be present inChlorella, had no effect on the phosphofructokinase.Chlorellaappeared to contain only one form of phosphofructokinase, possibly in the chloroplast. No pyrophosphate :D-fructose-6-P 1-phospho transferase activity could be dete
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