Calvet V E, Gil G, Hegardt F G
Arch Biochem Biophys. 1985 Feb 1;236(2):753-65. doi: 10.1016/0003-9861(85)90681-2.
Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature.
已通过涉及二乙氨基乙基纤维素、氨己基琼脂糖-4B和生物凝胶A 1.5m色谱法的过程，从大鼠肝脏胞质溶胶中纯化出四种具有作用于羟甲基戊二酰（HMG）-辅酶A还原酶、磷酸化酶和糖原合酶能力的磷蛋白磷酸酶。蛋白磷酸酶II（分子量180,000）是主要的酶（68%），具有非常广泛的底物特异性，对三种底物表现出相似的活性。磷酸酶I1（分子量180,000）和I3（分子量250,000）分别仅占总活性的12%和15%，并且它们也能够使三种底物去磷酸化。相比之下，磷酸酶I2（分子量200,000）仅表现出磷酸化酶磷酸酶活性，对HMG-辅酶A还原酶和糖原合酶的去磷酸化能力微不足道。在室温下用乙醇处理后，所有磷酸酶的分子量都发生了变化；蛋白磷酸酶I2、I3和II的分子量变为35,000，而磷酸酶I1的分子量降至69,000。所有四种磷酸酶中的糖原合酶磷酸酶活性均降低。以HMG-辅酶A还原酶和磷酸化酶为底物时，磷酸酶I1的活性也降低。乙醇处理后，磷酸酶I2、I3和II的HMG-辅酶A还原酶磷酸酶和磷酸化酶磷酸酶活性增加。每种蛋白磷酸酶都表现出不同的最适pH值，该值会根据底物而变化。这四种磷酸酶在Mn2+和Mg2+存在下活性增加。一般来说，Mn2+是比Mg2+更好的激活剂，并且磷酸酶I1对这些阳离子的依赖性比任何其他磷酸酶都更强。在蛋白磷酸酶I1、I3和II的HMG-辅酶A还原酶磷酸酶和糖原合酶磷酸酶反应中，磷酸化酶是一种竞争性底物。HMG-辅酶A还原酶也能够与磷酸化酶和糖原合酶竞争磷酸酶活性。糖原合酶磷酸酶活性在低分子量形式中受到的抑制较小。已与文献中先前报道的其他蛋白磷酸酶进行了比较。
