DePaoli-Roach A A, Ahmad Z, Roach P J
J Biol Chem. 1981 Sep 10;256(17):8955-62.
A protein kinase (designated PC0.7 in DePaoli-Roach, A. A., Roach, P. J., and Larner, J. (1979) J. Biol. Chem. 254, 12062-12068) that phosphorylated both glycogen synthase and phosvitin, has been extensively purified from rabbit skeletal muscle, close to apparent homogeneity. The enzyme activity was associated with two polypeptides, alpha (Mr = 43,000) and beta (Mr = 25,000), present in approximately equimolar amounts. The apparent molecular weight of the enzyme was 180,000, as determined by gel filtration, and 130,000, as judged from sucrose density gradient sedimentation. Unless precautions were taken during the purification, the alpha polypeptide underwent degradation, probably as a result of protease action. The beta polypeptide itself could be phosphorylated upon incubation of the enzyme with ATP and Mg2+ but no significant change in activity accompanied this phosphorylation reaction. The protein kinase was effective in utilizing both ATP and GTP as phosphate donors, with apparent Km values of 13 microM and 20-35 microM, respectively. The apparent Km values for phosvitin and glycogen synthase were 15 microM and greater than 10 microM, respectively. PC0.7 phosphorylated glycogen synthase to a level of approximately 0.5 phosphate/subunit, with little inactivation of the glycogen synthase. Phosphorylation occurred predominantly in a 21,000-dalton cyanogen bromide fragment of glycogen synthase, the same fragment preferentially phosphorylated by cyclic AMP-dependent protein kinase. This phosphorylation was also located in an approximately 17,000-dalton COOH-terminal region of the glycogen synthase molecule that is removed by limited tryptic proteolysis. Phosphorylation of glycogen synthase by PC0.7 occurred at serine residues whereas in phosvitin both serine and threonine residues were modified by PC0.7 action.
一种可使糖原合酶和卵黄高磷蛋白磷酸化的蛋白激酶（在德保利 - 罗奇、A.A.、罗奇、P.J.和拉纳、J.（1979年）《生物化学杂志》254卷，12062 - 12068页中被命名为PC0.7）已从兔骨骼肌中得到了广泛纯化，接近表观均一性。该酶活性与两种多肽相关，α（Mr = 43,000）和β（Mr = 25,000），它们的含量大致相等。通过凝胶过滤测定，该酶的表观分子量为180,000，而根据蔗糖密度梯度沉降判断为130,000。除非在纯化过程中采取预防措施，α多肽会发生降解，可能是蛋白酶作用的结果。β多肽本身在酶与ATP和Mg2 +一起温育时可被磷酸化，但这种磷酸化反应并未伴随活性的显著变化。该蛋白激酶能够有效地利用ATP和GTP作为磷酸供体，其表观Km值分别为13 microM和20 - 35 microM。卵黄高磷蛋白和糖原合酶的表观Km值分别为15 microM和大于10 microM。PC0.7将糖原合酶磷酸化至约0.5个磷酸/亚基的水平，糖原合酶几乎没有失活。磷酸化主要发生在糖原合酶的一个21,000道尔顿的溴化氰片段中，该片段也是环腺苷酸依赖性蛋白激酶优先磷酸化的片段。这种磷酸化也位于糖原合酶分子中一个约17,000道尔顿的COOH末端区域，该区域可通过有限的胰蛋白酶消化去除。PC0.7使糖原合酶磷酸化发生在丝氨酸残基上，而在卵黄高磷蛋白中，丝氨酸和苏氨酸残基都因PC0.7的作用而被修饰。
