Ref ID : 4324
Eckhard Werries and Ina Thurn; Breakdown of Glucopolysaccharides in Entamoeba histolytica by Phosphorylase. J.Protozool. 36(6):607-612, 1989
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Homogenates of trophozoites of Entamoeba histolytica released glucose 1-phosphate from amylopectin, glycogen, and amylose is a ratio of 100:78:74 at glucopolysaccharide concentrations of 0.1%. By use of self-generating Percoll gradients this activity was shown to be particulate and associated with glycogen. The phosphorylase was extracted from the 40,000 g pellet in aqueous medium and purified to homogeneity by gel filtration of Fractogel TSK HW-55(F) followed by chromatography on Blue Sepharose CL-6B. The purified enzyme was active not only against the glucopolysaccharides but also on dextrins with more than 3 glucose moieties, which were primarily formed by the action of amoebic amylases. At substrate concentrations of 1mM nonreducing ends of each glucan, the phosphorolysis rate of the branched polysaccharides was about 1.75x10E4 times higher than those of the maltodextrins. By means of HPLC the sequential degradation of 4-nitrophenyl-maltoheptaoside (G7-pNP) was studied. Native phosphorylase exhibited a relative molecular mass of Mr=200,000 by gel filtration and gel electophoresis. The SDS electrophoresis, under reducing conditions, indicated that the native enzyme was a dimer. Optimal degradation of the polysaccharides and dextrins was achieved at pH values of 7.5 and 7.0, respectively.