Werries E, Franz A, Geisemeyer S
Fachbereich Biologie/Chemie, Universität Osnabrück, Abteilung Biochemie, Federal Republic of Germany.
J Protozool. 1990 Nov-Dec;37(6):576-80. doi: 10.1111/j.1550-7408.1990.tb01268.x.
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.
溶组织内阿米巴滋养体的匀浆可使糖原完全降解，而仅来自同一来源的纯化磷酸化酶产生的终产物是极限糊精。通过在水性介质中提取，从40,000g沉淀中获得了一种能够使极限糊精去分支的酶系统，通过在Fractogel TSK HW-55(F)上进行凝胶过滤纯化，并通过在Blue Sepharose CL-6B和氨基丁基琼脂糖上进行色谱分离与磷酸化酶分离。根据圆盘凝胶电泳标准，糖原去分支系统被纯化了540倍，达到了均一状态。纯化后的酶在磷酸化酶存在的情况下能够降解糖原-极限糊精，并表现出淀粉-1,6-葡萄糖苷酶（EC 3.2.1.33）和4-α-葡聚糖转移酶（EC 2.4.1.25）的活性。虽然淀粉-1,6-葡萄糖苷酶从糖原-磷酸化酶极限糊精中释放葡萄糖，但转移酶活性将单个葡萄糖残基从极限糊精转移到4-硝基苯基-α-葡萄糖苷，依次产生可通过高效液相色谱检测到的4-硝基苯基-α-麦芽糖苷和4-硝基苯基-α-麦芽三糖苷。通过凝胶过滤以及在变性和非变性条件下的凝胶电泳，天然糖原去分支系统的相对分子质量为Mr = 180,000 +/- 10%。
