THE PROTEIN PHOSPHATASES INVOLVED IN CELLULAR-REGULATION - COMPARISON OF NATIVE AND RECONSTITUTED MG-ATP-DEPENDENT PROTEIN PHOSPHATASES FROM RABBIT SKELETAL-MUSCLE
THE PROTEIN PHOSPHATASES INVOLVED IN CELLULAR-REGULATION - COMPARISON OF NATIVE AND RECONSTITUTED MG-ATP-DEPENDENT PROTEIN PHOSPHATASES FROM RABBIT SKELETAL-MUSCLE
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DOI：
10.1111/j.1432-1033.1984.tb08521.x
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
COHEN, P
中科院分区：
文献类型：
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作者：
TUNG, HYL;COHEN, P
通过避免使用有机溶剂或在90-100的加热的程序，从兔骨骼肌中分离出天然Mg-ATP依赖性蛋白磷酸酶。 C.纯化的酶由以1：1摩尔比的2种主要蛋白质（分子量为37 kDa [kilodaltons]和31 kDa）组成，占材料的70-80％。与蛋白质磷酸酶-1的催化亚基合作的37-kDa成分，其与该蛋白质的身份是通过肽映射建立的，并通过其对特征性的34 kDa和33-kDa片段的裂解来确定，并在与挥发蛋白酶孵育后建立。与抑制剂-2混合的31 kDA蛋白质，其与该蛋白的认同是通过其热稳定性，在纳摩尔浓度下抑制蛋白质磷酸酶-1的能力以及通过糖源合酶激酶3对苏氨酸残基抑制蛋白质磷酸酶-1的能力。天然MG-ATP依赖性蛋白磷酸酶可能由蛋白质的催化亚基组成磷酸酶-1（37 kDa）和抑制剂-2（31 kDa）以1：1摩尔比。天然MG-ATP依赖性蛋白磷酸酶实际上具有与从抑制剂-2重构的酶和蛋白磷酸酶-1的37 kDa催化亚基相同的特性。每种制剂具有相似的特异性活性，并被相同浓度的抑制剂1所抑制。可以通过与糖原合酶激酶3和MG-ATP或MN2+和胰蛋白酶（或胰胆红素）孵育来激活这两种酶。但是，单独使用MN2+或在没有MN2+的情况下蛋白酶消化未能激活任何一种制剂。与糖原合酶激酶3和Mg-ATP孵育不会解离本地或重建的酶，而用MN2+和胰蛋白酶处理的酶从70 kDa降低至35 kDa。与胰凝乳蛋白酶一起孵育，将天然和重建的酶转化为需要与糖原合酶激酶3，MG-ATP和抑制剂-2预孵育的形式，以表现出催化活性。与从未依赖的37 kDa催化亚基的酶相反，与激活后分离的33 kDa催化亚基解离的33 kDa催化亚基的MG-ATP蛋白磷酸酶重构。在多肽的1末期，一个3至4 kDa片段可能参与加强未基因的37 kDa催化亚基与抑制剂-2的磷酸化形式之间的相互作用。
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.degree. C. The purified enzyme was composed of 2 major proteins (molecular mass 37 kDa [kilodaltons] 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. Therefore, the native Mg-ATP-dependent protein phosphatase is probably 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, 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. A 3- to 4-kDa fragment at 1 end of the polypeptide may be involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.