Tirard A, Roques M
Biochim Biophys Acta. 1978 Dec 20;537(2):485-94. doi: 10.1016/0005-2795(78)90533-0.
Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.
通过在Bio Gel 1.5m上进行凝胶过滤，随后在DEAE Sephadex A25上进行离子交换色谱，对猪甲状腺细胞质中的环磷酸腺苷结合蛋白进行了部分纯化。通过凝胶过滤分离出了具有环磷酸腺苷结合活性的三个组分（I、II、III）。近似分子量分别为280000、145000和65000。组分I在DEAE-Sephadex A25上进一步分离为两个峰（Iα和Iβ）。组分I、Iα、Iβ与蛋白激酶活性共迁移，而峰II和III则没有。这些组分在以下特征方面存在差异：环磷酸腺苷与分离组分的结合速率和稳定性受pH（4.0或7.5）的影响不同。用环[3H]AMP预孵育的组分在5%聚丙烯酰胺凝胶上的电泳迁移率显示，Iα、Iβ或II的迁移率相似（Rf 0.37），而组分III的迁移率则大得多（RF 0.73）；Scatchard图对于组分Iα、II和III呈线性，表观Kd在相同范围内（2至5 nM），而组分Iβ产生双相图，Kd为0.4 nM和20 nM；如Sephadex G 150过滤所示，添加到组分I、Iα或Iβ中的环[3H]AMP产生了分子量较低的环[3H]AMP结合蛋白复合物；根据洗脱体积，该复合物与组分II无法区分。在这项工作过程中，我们在纯化的第一步（Bio Gel 1.5m）分离出了一种与环磷酸腺苷结合活性无关的蛋白激酶，与组蛋白II A相比，它对鱼精蛋白表现出明显的特异性。
