Picton C, Klee C B, Cohen P
Eur J Biochem. 1980 Oct;111(2):553-61. doi: 10.1111/j.1432-1033.1980.tb04971.x.
Phosphorylase kinase has the structure (alpha beta gamma delta)4 where the delta-subunit is identical to the calcium-binding protein termed calmodulin [Shenolikar et al. (1979) Eur. J. Biochem. 100, 329--337]. The delta-subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The delta-subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the delta-subunit was associated with the gamma-subunit. This finding was confirmed by cross-linking experiments with dimethylsuberimidate which resulted in the formation of a gamma delta complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per alpha beta gamma delta unit, termed the delta'-subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the delta'-subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 microM. This was similar to the concentration of delta'-subunit which produced half-maximal activation. The delta'-subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the delta-subunit and [14C]calmodulin, and the calcium-dependent binding of the delta'-subunit allowed cross-linking experiments to be used which demonstrated that the delta'-subunit was bound to both the alpha and beta subunits. This result was supported by the finding that selective proteolysis of either the alpha-subunit, or the alpha and beta subunits, decreased or abolished the ability of phosphorylase kinase to bind to calmodulin-Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.
磷酸化酶激酶具有(αβγδ)4结构，其中δ亚基与称为钙调蛋白的钙结合蛋白相同[Shenolikar等人(1979年)，《欧洲生物化学杂志》100, 329 - 337]。在没有钙离子的情况下，δ亚基与磷酸化酶激酶紧密结合，其与[14C]钙调蛋白的交换速率仅为每周15%。只要存在钙离子，δ亚基在8 M尿素存在的情况下仍与磷酸化酶激酶结合，这一特性使得能够使用电泳技术，结果表明δ亚基与γ亚基相关联。用亚胺二甲酯进行的交联实验证实了这一发现，该实验导致形成了γδ复合物。结果表明，每个αβγδ单元的磷酸化酶激酶还结合一个额外的钙调蛋白分子，称为δ'亚基。在[14C]钙调蛋白存在下进行甘油梯度离心表明，δ'亚基与磷酸化酶激酶的相互作用仅在钙离子存在时发生，其解离常数(Kd)值接近0.01 μM。这与产生最大激活一半时的δ'亚基浓度相似。在8 M尿素存在的情况下，无论有无钙离子，δ'亚基都不会与磷酸化酶激酶保持结合。δ亚基与[14C]钙调蛋白之间非常缓慢的交换以及δ'亚基的钙依赖性结合使得能够使用交联实验，结果表明δ'亚基与α亚基和β亚基都结合。α亚基或α亚基和β亚基的选择性蛋白水解会降低或消除磷酸化酶激酶与钙调蛋白琼脂糖结合的能力，这一发现支持了该结果。文中讨论了不同亚基在磷酸化酶激酶活性调节中的作用。
