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The World of Protozoa, Rotifera, Nematoda and Oligochaeta

Ref ID : 4307

Eckhard Werries, Alfred Franz, and Sibylle Geisemeyer; Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica. J.Protozool. 37(6):576-580, 1990

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Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (E.C. 3.2.1.33) and 4-alpha-glucanotransferase (E.C. 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr=180,000+/-10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.