Fox J, Kawaguchi K, Greenberg E, Preiss J
Biochemistry. 1976 Feb 24;15(4):849-57. doi: 10.1021/bi00649a019.
The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained: glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity. One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000. The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000. Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction. The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3 kcal.
利用4-氨丁基琼脂糖柱已将大肠杆菌B糖原合酶纯化至表观均一。得到了该酶的两个组分：比活性为380 μmol mg-1且无分支酶活性的糖原合酶I，以及比活性为505 μmol mg-1且含有分支酶活性（其为糖原合酶所观察到活性的0.1%）的糖原合酶II。在圆盘凝胶电泳中，每个糖原合酶组分仅发现一条蛋白带，且它们与糖原合酶活性一致。在糖原合酶组分的十二烷基硫酸钠凝胶电泳中观察到一条主要蛋白带和一条几乎未进入凝胶的非常淡的蛋白带。在两种糖原合酶组分的十二烷基硫酸钠凝胶电泳中，主要蛋白带的亚基分子量测定为49 000±2 000。通过蔗糖密度梯度超速离心法测定了天然酶的分子量。糖原合酶I的分子量为93 000，而糖原合酶II的分子量为200 000。在4℃或-85℃下放置时，两种酶均转变为分子量为98 000、135 000和185 000的形式。因此，大肠杆菌B糖原合酶的活性形式可以以亚基的二聚体、三聚体和四聚体形式存在。该酶被证明可催化葡萄糖从ADP葡萄糖转移至麦芽糖以及麦芽糊精系列的更高寡糖，但不能转移至葡萄糖。已证明1,5-葡萄糖酸内酯是糖原合酶反应的有效抑制剂。已证明糖原合酶反应是可逆的。标记的ADP葡萄糖可由[14C]ADP或[14C]糖原形成。测定了37℃平衡时ADP与ADP葡萄糖的比率，发现在5.27 - 6. .82的pH范围内变化三倍。根据这些数据，平衡时ADP2-与ADP葡萄糖的比率测定为45.8±4.5。假设α-1,4-糖苷键水解的ΔF°为-4.0千卡，则ADP葡萄糖中糖苷键水解的ΔF°为-6.3千卡。
