Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica.
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 (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 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.
Werries
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Fachbereich Biologie/Chemie, Universität Osnabrück, Abteilung Biochemie, Federal Republic of Germany.
Franz
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Geisemeyer
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D000818:Animals; D002384:Catalysis; D004748:Entamoeba histolytica / Q000201:enzymology*; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000032:analysis* / Q000378:metabolism; D010762:Phosphorylase a / Q000032:analysis
D006004:Glycogen Debranching Enzyme System; D006003:Glycogen; D010762:Phosphorylase a