Yeaman S J, Cohen P
Eur J Biochem. 1975 Feb 3;51(1):93-104. doi: 10.1111/j.1432-1033.1975.tb03910.x.
Phosphorylase kinase was activated 5--10-fold in vivo by an intravenous injection of adrenalin. Sodium fluoride an inhibitor of phosphorylase kinase phosphatase, was required to prevent the reversal of this process; the activated and non-activated forms of the enzyme were indistinguishable by dodecylsulphate gel electrophoresis. This suggested that the activation had resulted from a phosphorylation of the enzyme, and that it was not a consequence of the well known activation by proteolytic cleavage that can be demonstrated in vitro. Phosphorylase kinase activated in vivo was purified and digested with trypsin, and the two tryptic peptides which contain the serine residues which are phosphorylated in vitro by the action of cyclic-AMP (adenosine 3':5'-monophosphate) dependent protein kinase, were isolated. It was found that the same nine-amino-acid segment of the beta chain and the same seven-amino-acid segment of the alpha chain had become phosphorylated in vivo in response to adrenalin, as were phosphorylated in vitro. The degree of phosphorylation of each of the two sites was at least 50%. The data provide direct proof that the activation of phosphorylase kinase which occurs in vivo in response to adrenalin results from a phosphorylation of the enzyme. They also indicate that the novel form of regulation associated with the phosphorylation of the alpha subunit, the stimulation of protein dephosphorylation by "second site phosphorylation", can now be regarded as a new form of enzyme control mechanism which operates in vivo. The regulation of phosphorylase kinase activity was studied in the protein - glycogen complex from skeletal muscle. The enzyme could be rapidly converted to a phosphorylated form in a cyclic-AMP-stimulated reaction upon addition of magnesium ions and ATP, but the conversion of phosphorylase b to phosphorylase a in the complex still showed an absolute requirement for calcium ions. The implications of these findings and major problems in the hormonal control of skeletal muscle glycogenolysis which are not yet resolved, are discussed.
静脉注射肾上腺素可使磷酸化酶激酶在体内被激活5至10倍。需要用氟化钠（一种磷酸化酶激酶磷酸酶的抑制剂）来防止这一过程的逆转；通过十二烷基硫酸钠凝胶电泳无法区分该酶的激活形式和未激活形式。这表明激活是由该酶的磷酸化引起的，而不是体外可证明的通过蛋白水解裂解进行的众所周知的激活的结果。对体内激活的磷酸化酶激酶进行纯化并用胰蛋白酶消化，分离出两条含有丝氨酸残基的胰蛋白酶肽段，这些丝氨酸残基在体外可被环磷酸腺苷（腺苷3':5'-单磷酸）依赖性蛋白激酶作用而磷酸化。结果发现，β链的相同九氨基酸片段和α链的相同七氨基酸片段在体内因肾上腺素而发生了磷酸化，与体外磷酸化情况相同。两个位点的磷酸化程度至少为50%。这些数据直接证明了体内因肾上腺素而发生的磷酸化酶激酶的激活是由该酶的磷酸化引起的。它们还表明，与α亚基磷酸化相关的新型调节形式，即“第二位点磷酸化”对蛋白质去磷酸化的刺激作用，现在可被视为一种在体内起作用的新的酶控制机制。在骨骼肌的蛋白质 - 糖原复合物中研究了磷酸化酶激酶活性的调节。加入镁离子和ATP后，在环磷酸腺苷刺激的反应中，该酶可迅速转化为磷酸化形式，但复合物中磷酸化酶b向磷酸化酶a的转化仍然绝对需要钙离子。讨论了这些发现的意义以及骨骼肌糖原分解激素控制中尚未解决的主要问题。
