Brautigan D L, Shriner C L, Gruppuso P A
J Biol Chem. 1985 Apr 10;260(7):4295-302.
An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.
一种分子量为33,000的磷酸化酶磷酸酶活性形式，在十多年里一直被称为催化亚基，从兔骨骼肌中纯化至近乎均一。用该磷酸酶对绵羊进行反复免疫，产生了能阻断磷酸酶活性的免疫球蛋白。这些免疫球蛋白在固定化磷酸化酶磷酸酶柱上进行亲和纯化，并在与过氧化物酶偶联的兔抗绵羊免疫球蛋白的“Western”免疫印迹法中用作大分子探针。在免疫原样品中，只有一种分子量为33,000的蛋白质被染色，证明了探针的特异性。然而，在肌肉提取物或部分纯化制剂中未检测到分子量为33,000的磷酸酶蛋白。相反，检测到一种分子量为70,000的单一蛋白质。有限的蛋白水解作用，特别是由金黄色葡萄球菌V8蛋白酶和嗜热菌蛋白酶引起的，将免疫反应性蛋白从分子量70,000转化为分子量33,000。在室温下用80%乙醇使磷酸酶制剂凝固，使分子量70,000的蛋白质不溶，但能从沉淀中提取出分子量33,000的蛋白质。因此，我们得出结论，分子量70,000的免疫反应性蛋白是磷酸化酶磷酸酶的“催化亚基”，其催化结构域分子量为33,000。由于其相对抗蛋白水解和凝固作用，以前的纯化方案只得到了分子量33,000的片段。磷酸化酶磷酸酶“天然”形式的凝胶过滤色谱显示分子量约为230,000。在经Co2+和胰蛋白酶处理后表现出活性的相同级分中，通过免疫印迹法检测到分子量70,000的催化亚基和与抑制剂-2相关的分子量60,000的蛋白质。在此激活过程中，只有分子量60,000的蛋白质被降解。我们提出，天然磷酸化酶磷酸酶是一种具有双重对称性的细长结构，包含一个分子量70,000的催化亚基和一个分子量60,000的调节亚基。
