Breakdown of glucopolysaccharides in Entamoeba histolytica by phosphorylase.
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 x 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.
Werries
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Fachbereich Biologie/Chemie der Universität Osnabrück, Federal Republic of Germany.
Thurn
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D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000378:metabolism; D006868:Hydrolysis; D006005:Phosphorylases / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*
D011134:Polysaccharides; D006005:Phosphorylases