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Breakdown of Glucopolysaccharides in Entamoeba histolytica by Phosphorylase
Author(s) -
WERRIES ECKHARD,
THURN INA
Publication year - 1989
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1989.tb01103.x
Subject(s) - amylopectin , glycogen phosphorylase , chromatography , chemistry , amylose , size exclusion chromatography , phosphorolysis , polysaccharide , starch , entamoeba histolytica , maltose , biochemistry , glucan , enzyme , biology , microbiology and biotechnology , purine nucleoside phosphorylase , purine
Homogenates of trophozoites of Entamoeba histolytica released glucose 1‐phosphate from amylopectin, glycogen, and amylose in 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 on 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 1 mM nonreducing ends of each glucan, the phosphorolysis rate of the branched polysaccharides was about 1.75 × 10 4 times higher than those of the maltodextrins. By means of HPLC the sequential degradation of 4‐nitrophenyl‐maltoheptaoside (G 7 ‐pNP) was studied. Native phosphorylase exhibited a relative molecular mass of M r = 200,000 by gel filtration and gel electrophoresis. 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.