Bywater R, Gustavsson J G
J Biochem Biophys Methods. 1980 Jan-Feb;2(1):49-61. doi: 10.1016/0165-022x(80)90073-1.
A novel synthesis route for the preparation of a cyclic AMP-containing affinity adsorbent is described. This involves the use of a simple single-vessel, two-step, solid-phase reaction in which 5'-AMP, originally coupled to the matrix in the form of 5'-AMP-Sepharose 4B, is cyclised by a sequence involving activation of the phosphate moiety, followed by intramolecular condensation to give a 3',5'-phosphodiester. Physicochemical evidence, including 31P NMR studies, shows that this cyclisation takes place leaving no trace of original phosphomonoesters. The gel retains its excellent chromatographic properties, but now its specificity is directed towards cAMP-dependent proteins. Protein kinase (EC 2.7.1.37) binds to the gel, the catalytic subunit only being eluted when a simple buffer 0.2 M phosphate/1 mM theophylline is applied to column, whilst the regulatory unit is eluted using 20% ethylene glycol of 2% Pharmalyte, pH 5-8. This gel should find a use in the purification of protein kinase subunits and, by inference, of other cAMP-dependent proteins.
描述了一种制备含环磷酸腺苷亲和吸附剂的新合成路线。这涉及使用简单的单容器两步固相反应，其中最初以5'-腺苷酸-琼脂糖4B形式偶联到基质上的5'-腺苷酸通过一系列反应环化，该系列反应包括磷酸部分的活化，随后进行分子内缩合以生成3',5'-磷酸二酯。包括31P NMR研究在内的物理化学证据表明，这种环化反应发生后没有留下原始磷酸单酯的痕迹。该凝胶保留了其优异的色谱性能，但现在其特异性针对环磷酸腺苷依赖性蛋白。蛋白激酶（EC 2.7.1.37）与凝胶结合，只有当向柱中加入0.2 M磷酸盐/1 mM茶碱的简单缓冲液时，催化亚基才会被洗脱，而调节亚基则使用20%乙二醇或2%两性电解质（pH 5-8）洗脱。这种凝胶可用于纯化蛋白激酶亚基，由此推断，也可用于纯化其他环磷酸腺苷依赖性蛋白。
