Bollen M, Vandenheede J R, Goris J, Stalmans W
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Biochim Biophys Acta. 1988 Apr 2;969(1):66-77. doi: 10.1016/0167-4889(88)90089-4.
Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.
对大鼠肝脏的线粒体后上清液进行分级分离时，合酶磷酸酶（EC 3.1.3.42）活性（在高组织浓度下测定）大部分在糖原级分中回收，少量在胞质溶胶中回收。相比之下，磷酸化酶磷酸酶（EC 3.1.3.17）活性在这两个级分中大致均匀分布，在微粒体级分中回收的量较少。微粒体和糖原级分中的磷酸酶活性在与调节蛋白（一种1型（ATP、Mg依赖性）蛋白磷酸酶的特异性抑制剂）预孵育后几乎完全被抑制。然而，在胞质级分中，2A型（多阳离子刺激型）磷酸酶对磷酸化酶和体外磷酸化的肌肉合酶的去磷酸化有显著贡献。在整个这项工作中用作合酶磷酸酶测量底物的肝脏合酶b仅被调节蛋白敏感的磷酸酶激活。对亚细胞级分进行胰蛋白酶处理导致对调节蛋白的敏感性显著增加（高达1000倍），磷酸化酶磷酸酶活性增加几倍，合酶磷酸酶活性完全丧失。在稀释与糖原结合的酶时也发生了类似的变化。与已知可抵消抑制剂-1和调节蛋白作用的去抑制剂蛋白预孵育，可使磷酸化酶磷酸酶活性增加几倍，但仅在胞质和微粒体级分中。它不影响合酶磷酸酶活性。综上所述，结果表明在胞质、微粒体和糖原级分中存在不同的多亚基1型磷酸酶。
