Andreeva I E, Livanova N B, Eronina T B, Silonova G V, Poglazov B F
Eur J Biochem. 1986 Jul 1;158(1):99-106. doi: 10.1111/j.1432-1033.1986.tb09726.x.
Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.
通过十二烷基硫酸钠（SDS）凝胶电泳判断，磷酸化酶激酶已从白色和红色鸡骨骼肌中纯化至接近均一。通过琼脂糖4B柱层析估计，天然酶的分子量与兔骨骼肌磷酸化酶激酶相似，即1320 kDa。在SDS存在下进行聚丙烯酰胺凝胶电泳时，从白色和红色肌肉中纯化得到的酶均显示有四个亚基，分别对应分子量为140 kDa、129 kDa、44 kDa和17 kDa的α'、β、γ'和δ亚基。根据天然酶1320 kDa的分子量以及从聚丙烯酰胺凝胶的光密度扫描图估算的亚基摩尔比，提出了一个亚基组成公式(α'βγ'δ)4。针对鸡磷酸化酶激酶α'和β亚基混合物的抗血清与鸡酶产生单一沉淀线，但与兔骨骼肌磷酸化酶激酶无交叉反应。不同制备的鸡骨骼肌磷酸化酶激酶的pH 6.8/8.2活性比在0.3至0.5之间变化。鸡磷酸化酶激酶可将兔磷酸化酶b用作底物，在pH 8.2时表观Km值为0.02 mM。在pH 8.2（0.20 mM）时，ATP的表观V（18 μmol min-1 mg-1）和Km值与纯化的兔磷酸化酶激酶b处于同一数量级。鸡磷酸化酶激酶的活性很大程度上依赖于Ca2+。鸡酶被钙调蛋白和肌钙蛋白C激活2至4倍，半最大激活浓度分别为2 nM和0.1 μM。用依赖cAMP的蛋白激酶催化亚基进行磷酸化（高达2 mol 32P/mol αβγδ单体）和自身磷酸化（高达8 mol 32P/mol αβγδ单体）分别使活性增加1.5倍和2倍。对鸡磷酸化酶激酶进行有限的胰蛋白酶和胰凝乳蛋白酶水解可使其活性提高2倍。对蛋白水解攻击产物的电泳分析表明，兔和鸡γ亚基的结构存在一些差异，兔红色肌肉和鸡α'亚基的结构存在一些相似性。
