Antoniw J F, Nimmo H G, Yeaman S J, Cohen P
Biochem J. 1977 Feb 15;162(2):423-33. doi: 10.1042/bj1620423.
Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.
将肌肉提取物用乙醇分级分离，在DEAE - 纤维素上进行层析，用硫酸铵沉淀，并用葡聚糖G - 200进行凝胶过滤。使用以下七种磷酸化蛋白质底物测定这些级分的蛋白质磷酸酶活性：磷酸化酶a、糖原合酶b1、糖原合酶b2、磷酸化酶激酶（α亚基或β亚基磷酸化）、组蛋白H1和组蛋白H2B。通过最后的凝胶过滤步骤分离出三种具有独特特异性的蛋白质磷酸酶，分别称为I、II和III。蛋白质磷酸酶 - I，表观分子量为300000，是一种活性组蛋白磷酸酶，但它仅占从葡聚糖G - 200柱中回收的糖原合酶磷酸酶 - 1和糖原合酶磷酸酶 - 2活性的10 - 15%，以及磷酸化酶激酶磷酸酶和磷酸化酶磷酸酶活性的2 - 3%。蛋白质磷酸酶 - II，表观分子量为170000，具有与蛋白质磷酸酶 - I相似的组蛋白磷酸酶活性。它具有从葡聚糖G - 200中回收的针对磷酸化酶激酶α亚基活性的95%以上。它占糖原合酶磷酸酶 - 1和糖原合酶磷酸酶 - 2活性的10 - 15%，但占针对磷酸化酶激酶β亚基活性的不到5%，以及从葡聚糖G - 200中回收的磷酸化酶磷酸酶活性的1 - 2%。蛋白质磷酸酶 - III是活性最高的组蛋白磷酸酶。它具有从葡聚糖G - 200中回收的95%的磷酸化酶磷酸酶和β - 磷酸化酶激酶磷酸酶活性，以及75%的糖原合酶磷酸酶 - 1和糖原合酶磷酸酶 - 2活性。它占α - 磷酸化酶激酶磷酸酶活性的不到5%。蛋白质磷酸酶 - III有时以表观分子量为75000的形式（称为IIIA）从葡聚糖G - 200中洗脱，有时以分子量为46000的形式（称为IIIB）洗脱，有时以两种组分的混合物形式洗脱。蛋白质磷酸酶 - IIA和 - IIB的底物特异性相同。这些发现，结合磷酸化酶磷酸酶、β - 磷酸化酶激酶磷酸酶、糖原合酶磷酸酶 - 1和糖原合酶磷酸酶 - 2活性在葡聚糖G - 200步骤之前共纯化的观察结果，表明单一的蛋白质磷酸酶（蛋白质磷酸酶 - III）催化抑制糖原分解或刺激糖原合成的每个去磷酸化反应。以下论文[科恩，P.，尼莫，G.A.和安东尼，J.F.（1977年）《生物化学杂志》1628 435 - 444]中描述的一种热稳定蛋白质是蛋白质磷酸酶 - III的特异性抑制剂的结果进一步支持了这一论点。
