Mitchell J W, Mellgren R L, Thomas J A
J Biol Chem. 1980 Nov 10;255(21):10368-74.
Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.
从牛心脏中纯化得到的糖原合酶I，比活性为33单位/毫克，在十二烷基硫酸钠凝胶电泳上呈现单一条带，亚基分子量为86,000。该酶用同样从心脏中分离得到的cAMP依赖性蛋白激酶催化亚基进行磷酸化。在有10微摩尔ATP的情况下，每个糖原合酶亚基仅掺入一个磷酸基团。当用硫酸钠和6-磷酸葡萄糖进行测定时，磷酸化使糖原合酶I的活性百分比从0.95降至0.50。每个亚基含有一个磷酸基团的糖原合酶被命名为GS-1。当ATP浓度增加到0.5毫摩尔时，每个合酶亚基又额外掺入一个磷酸基团，糖原合酶I的百分比从0.50降至<0.05。这种酶形式被命名为GS-1,2。GS-1向Gs-1,2的转化呈现出与ATP浓度相关的协同动力学，在约40微摩尔时达到半最大刺激。GS-1的磷酸化也可通过加入其他非底物三磷酸核苷酸如ITP和UTP以及10微摩尔ATP来实现。6-磷酸葡萄糖和硫酸钠对该磷酸化反应无影响。用CNBr裂解32P标记的GS-1,2后得到两个单独的肽段，而GS-1仅得到一个。两种酶形式都含有一个共同的单磷酸化肽段。因此，心脏糖原合酶可能使用适当浓度的ATP在两个不同位点中的任意一个进行特异性磷酸化。ATP作为蛋白激酶的底物，同时也影响cAMP依赖性蛋白激酶对第二个磷酸化位点的可及性。
