Tung H Y, Cohen P
Eur J Biochem. 1984 Nov 15;145(1):57-64. doi: 10.1111/j.1432-1033.1984.tb08521.x.
The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
通过一种避免使用有机溶剂或在90 - 100摄氏度加热的方法，从兔骨骼肌中分离出了“天然的”Mg - ATP依赖性蛋白磷酸酶。纯化后的酶由两种主要蛋白质组成（分子量分别为37 kDa和31 kDa），二者以1:1的摩尔比存在，占总物质的70 - 80%。37 kDa的组分与蛋白磷酸酶 - 1的催化亚基迁移率相同，通过肽图谱分析以及与胰凝乳蛋白酶孵育后裂解为特征性的34 kDa和33 kDa片段，确定了其与该蛋白的一致性。31 kDa的蛋白质与抑制剂 - 2迁移率相同，通过其热稳定性、在纳摩尔浓度下抑制蛋白磷酸酶 - 1的能力以及被糖原合酶激酶3磷酸化的苏氨酸残基，确定了其与该蛋白的一致性。因此得出结论，“天然的”Mg - ATP依赖性蛋白磷酸酶由蛋白磷酸酶 - 1的催化亚基（37 kDa）和抑制剂 - 2（31 kDa）以1:1的摩尔比组成。“天然的”Mg - ATP依赖性蛋白磷酸酶与由抑制剂 - 2和蛋白磷酸酶 - 1的37 kDa催化亚基重构的酶具有几乎相同的性质。每种制剂具有相似的比活性，并且被相同浓度的抑制剂 - 1抑制。两种酶都可以通过与糖原合酶激酶 - 3和Mg - ATP孵育，或通过Mn2 +和胰蛋白酶（或胰凝乳蛋白酶）激活。然而，单独的Mn2 +或在没有Mn2 +的情况下进行蛋白酶消化，均不能激活任何一种制剂。与糖原合酶激酶 - 3和Mg - ATP孵育不会使“天然的”或“重构的”酶解离，而用Mn2 +和胰蛋白酶处理会使它们的表观分子量从70 kDa降至35 kDa。与胰凝乳蛋白酶孵育会将“天然的”和“重构的”酶转化为需要与糖原合酶激酶 - 3、Mg - ATP和抑制剂 - 2预孵育才能表现出催化活性的形式。与由“切口的”33 kDa催化亚基重构的Mg - ATP依赖性蛋白磷酸酶不同，由未降解的37 kDa催化亚基重构的酶在激活时会解离。结果表明，多肽一端的3 - 4 kDa片段参与加强未降解的37 kDa催化亚基与磷酸化形式的抑制剂 - 2之间的相互作用。
