Resink T J, Hemmings B A, Tung H Y, Cohen P
Eur J Biochem. 1983 Jun 15;133(2):455-61. doi: 10.1111/j.1432-1033.1983.tb07485.x.
Homogenous preparations of the catalytic subunit of protein phosphatase-1 and inhibitor-2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg-ATP-dependent protein phosphatase in that preincubation with glycogen synthase kinase-3 and Mg-ATP is required to generate activity. Activation results from the phosphorylation of inhibitor-2. The molar concentrations of protein phosphatase-1 and inhibitor-2 in rabbit skeletal muscle (0.25-0.5 microM) are similar. Incubation of the reconstituted Mg-ATP-dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor-2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase-3 and Mg-ATP provided that inhibitor-2 is added. This exogenous inhibitor-2 appears to displace the fragments of inhibitor-2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S.D., Vandenheede, J.R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231-10234] that inhibitor-2 can function as an 'activator' as well as an inhibitor of the Mg-ATP-dependent protein phosphatase. Incubation of the catalytic subunit of protein phosphatase-1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive form that could be partially reactivated by manganese ions, but not by glycogen synthase kinase-3 and Mg-ATP. Conversely, the reconstituted Mg-ATP-dependent protein phosphatase could only be activated by glycogen synthase kinase-3 and Mg-ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase-1 to a manganese-ion dependent form is a quite separate phenomenon from the formation of the Mg-ATP-dependent protein phosphatase. Inhibitor-2 can inactivate protein phosphatase-1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase-3 and Mg-ATP. This occurs at higher concentrations of inhibitor-2 than those required to form the Mg-ATP-dependent protein phosphatase, and appears to result from the binding of inhibitor-2 to a distinct site on the enzyme.
蛋白磷酸酶 -1催化亚基和抑制剂 -2的均一制剂可以结合产生一种无活性的酶，该酶由这两种蛋白质之间1:1的复合物组成。该物种与Mg-ATP依赖性蛋白磷酸酶无法区分，因为需要与糖原合酶激酶 -3和Mg-ATP预孵育才能产生活性。激活是由抑制剂 -2的磷酸化引起的。兔骨骼肌中蛋白磷酸酶 -1和抑制剂 -2的摩尔浓度（0.25 - 0.5 microM）相似。用胰凝乳蛋白酶孵育重组的Mg-ATP依赖性蛋白磷酸酶会伴随着抑制剂 -2的有限蛋白水解及其磷酸化位点的丧失。只要添加抑制剂 -2，该物种就可以被糖原合酶激酶 -3和Mg-ATP激活。这种外源性抑制剂 -2似乎从胰凝乳蛋白酶消化产生的酶中取代了抑制剂 -2的片段。这些实验可能解释了[Yang, S.D., Vandenheede, J.R.和Merlevede, W. (1981) J. Biol. Chem. 256, 10231 - 10234]的报告，即抑制剂 -2既可以作为Mg-ATP依赖性蛋白磷酸酶的“激活剂”，也可以作为其抑制剂。用氟化钠或焦磷酸钠孵育蛋白磷酸酶 -1的催化亚基会将该酶转化为无活性形式，这种形式可以被锰离子部分重新激活，但不能被糖原合酶激酶 -3和Mg-ATP激活。相反，重组的Mg-ATP依赖性蛋白磷酸酶只能被糖原合酶激酶 -3和Mg-ATP激活，而不能被锰离子激活。得出的结论是，蛋白磷酸酶 -1向锰离子依赖性形式的转化与Mg-ATP依赖性蛋白磷酸酶的形成是完全不同的现象。抑制剂 -2可以通过第二种机制使蛋白磷酸酶 -1失活，这种机制不能通过与糖原合酶激酶 -3和Mg-ATP预孵育来逆转。这发生在比形成Mg-ATP依赖性蛋白磷酸酶所需浓度更高 的抑制剂 -2浓度下，并且似乎是由于抑制剂 -2与酶上一个不同的位点结合所致。
