Rat skeletal muscle glycogen synthase: phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases.
Rat skeletal muscle glycogen synthase: phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases.
复制标题
大鼠骨骼肌糖原合酶：纯化酶被 cAMP 依赖性和非依赖性蛋白激酶磷酸化。
DOI：
10.1016/0003-9861(85)90606-x
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发表时间：
1985
影响因子：
3.9
通讯作者：
LawrenceJr,JC
中科院分区：
文献类型：
--
作者：
Hiken,JF;LawrenceJr,JC
糖原合酶从大鼠骨骼肌中纯化到接近同质，并发现在几个方面类似于兔骨骼肌酶。在十二烷基硫酸钠存在下，根据聚丙烯酰胺凝胶上亚基的电泳迁移率估计表观分子量（M app）为86，000。用胰蛋白酶对大鼠合酶进行有限的蛋白水解，导致形成M app s等于75，000、69，000和67，000的物种。该酶可被cAMP依赖性蛋白激酶、磷酸化酶激酶和cAMP非依赖性蛋白激酶PC 0.7和FA GSK-3磷酸化。基本上所有观察到的磷酸化都发生在位于两个溴化氰片段中的丝氨酸上，标记为CB-1（M app= 13，000）和CB-2（M app= 22，000）。F A两个片段中GSK-3和cAMP依赖性蛋白激酶磷酸化位点。由磷酸化酶激酶引入的磷酸盐仅位于CB-1中，并且在CB-2中发现与PC 0.7结合的磷酸盐。通过FA GSK-3的磷酸化降低了亚基的电泳迁移率，将异质性引入CB-2，并且与通过PC 0.7的磷酸化协同。为了更完全地分离磷酸化位点，糖原合酶的样品进行了广泛的蛋白水解，使用胰蛋白酶，然后通过反相液相色谱。当被相同的激酶磷酸化时，用大鼠和兔骨骼肌糖原合酶获得的片段的模式几乎相同。提出的结果提供了强有力的证据表明，大鼠骨骼肌糖原合酶的亚基至少有五个磷酸化位点，是非常相似的，如果不是相同的，目前对兔肌肉酶的网站。
Glycogen synthase was purified to near homogeneity from rat skeletal muscle, and was found to resemble the rabbit skeletal muscle enzyme in several respects. An apparent molecular weight (M app) of 86,000 was estimated from the electrophoretic mobility of the subunit on polyacrylamide gels in the presence of sodium dodecyl sulfate. Limited proteolysis of the rat synthase with trypsin resulted in the formation of species with M app s equal to 75,000, 69,000, and 67,000. The enzyme could be phosphorylated by cAMP-dependent protein kinase, phosphorylase kinase, and the cAMP-independent protein kinases, PC 0.7 and F A GSK-3. Essentially all of the phosphorylation observed occurred on serines located in two cyanogen bromide fragments, denoted CB-1 (M app= 13,000) and CB-2 (M app= 22,000). F A GSK-3 and cAMP-dependent protein kinase phosphorylated sites in both fragments. Phosphate introduced by phosphorylase kinase was located exclusively in CB-1, and that incorporated with PC 0.7 was found in CB-2. Phosphorylation by F A GSK-3 reduced the electrophoretic mobility of the subunit, introduced heterogeneity into CB-2, and was synergistic with phosphorylation by PC 0.7. To separate phosphorylation sites more completely, samples of glycogen synthase were subjected to extensive proteolysis using trypsin, followed by reverse-phase liquid chromatography. When phosphorylated by the same kinases, the pattern of fragments obtained with rat and rabbit skeletal muscle glycogen synthase were almost identical. The results presented provide strong evidence that the subunit of rat skeletal muscle glycogen synthase has at least five phosphorylation sites that are very similar, if not identical, to sites present on the rabbit muscle enzyme.