Purification and properties of rabbit skeletal muscle adenosine 3',5'-monophosphate-dependent protein kinases.
Purification and properties of rabbit skeletal muscle adenosine 3',5'-monophosphate-dependent protein kinases.
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兔骨骼肌腺苷 3,5-单磷酸依赖性蛋白激酶的纯化和特性。
DOI：
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发表时间：
1971
影响因子：
4.8
通讯作者：
E. Krebs
中科院分区：
文献类型：
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作者：
E. Reimann;D. Walsh;E. Krebs
摘要 描述了从兔骨骼肌中纯化腺苷 3',5'-单磷酸依赖性（环 AMP 依赖性）蛋白激酶的改进程序。该过程导致酶在二乙氨基乙基纤维素上分离成两个活性峰。没有观察到这两个峰的相互转化。两个峰都表现出对环 AMP 的依赖性，但在某些条件下，峰 I 对环 AMP 的依赖性可以降低。半最大刺激所需的环 AMP 浓度对于峰 I 为 3 x 10-8 m，对于峰 II 为 1.5 x 10-8 m。环 AMP 与蛋白激酶的结合可以通过多种温和的化学处理和 Sephadex G-25 上的凝胶过滤来逆转。对于峰 I 或峰 II，在存在或不存在环 AMP 的情况下，存在 10 mm Mg2+ 时 ATP 的表观 Km 约为 1.5 x 10-5 m。对于峰 II，在存在和不存在环 AMP 的情况下，酪蛋白的表观 Km 分别为 0.9 和 0.6 mg/ml。当反应混合物中存在 0.1 m NaCl 时，表观 Km 增加至 8 mg/ml。两种酶的酪蛋白最适 pH 值为 6.0，组蛋白磷酸化最适 pH 值为 6.5。峰 I 包含两个依赖于环 AMP 的组分，可通过蔗糖密度梯度离心分离；这些组分的沉降系数为 6.8 S 和 4.9 S。Peak II 活性以单峰形式沉积，沉降系数为 4.8 S。在环 AMP 存在下，Peak I 的蛋白激酶活性以单组分形式沉降，沉降系数为 3.4 S。在环 AMP 存在下，Peak II 的蛋白激酶活性也表现出比不存在该核苷酸时低得多的沉降系数。
Abstract A modified procedure for purification of the adenosine 3',5'-monophosphate-dependent (cyclic AMP-dependent) protein kinase from rabbit skeletal muscle is described. This procedure results in the separation of the enzyme into two peaks of activity on diethylaminoethylcellulose. No interconversion of these two peaks was observed. Both peaks exhibit dependence on cyclic AMP, but under certain conditions the dependence of Peak I on cyclic AMP can be reduced. The concentration of cyclic AMP needed for half-maximal stimulation is 3 x 10-8 m for Peak I and 1.5 x 10-8 m for Peak II. The binding of cyclic AMP to the protein kinase can be reversed by a number of mild chemical treatments and by gel filtration on Sephadex G-25. The apparent Km for ATP in the presence of 10 mm Mg2+ is approximately 1.5 x 10-5 m in the presence or absence of cyclic AMP for either Peak I or Peak II. For Peak II the apparent Km for casein is 0.9 and 0.6 mg per ml in the presence and absence of cyclic AMP, respectively. This apparent Km is increased to 8 mg per ml when 0.1 m NaCl is present in the reaction mixture. The pH optimum is 6.0 for casein and 6.5 for histone phosphorylation for both enzymes. Peak I contains two components which are dependent on cyclic AMP and separable by sucrose density gradient centrifugation; the sedimentation coefficients of these components are 6.8 S and 4.9 S. Peak II activity sediments as a single peak with a sedimentation coefficient of 4.8 S. In the presence of cyclic AMP the protein kinase activity of Peak I sediments as a single component with a sedimentation coefficient of 3.4 S. The protein kinase activity of Peak II also exhibits a much lower sedimentation coefficient in the presence of cyclic AMP than that shown in the absence of this nucleotide.