Døskeland S O, Haga H J
Biochem J. 1978 Aug 15;174(2):363-72. doi: 10.1042/bj1740363.
An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.
本文描述了一种环磷酸腺苷（cAMP）检测方法，该方法利用了环磷酸腺苷依赖性蛋白激酶I解离后的受体部分对核苷酸的高亲和力。通过盐（800 mM - NaCl/30 mM - EDTA）使激酶保持解离状态。在存在一种简单制备的热稳定蛋白组分的情况下，结合试剂在达到结合平衡所需的时间内保持稳定。本文还描述了一种简单的方法[用聚（乙二醇）沉淀，然后进行DEAE - 纤维素色谱法]，用于从兔骨骼肌中分离蛋白激酶I与其他环磷酸腺苷结合蛋白。该检测方法的灵敏度、精密度、重现性和特异性与其他环磷酸腺苷检测方法相比具有优势。本检测方法的主要优点是它对多种盐、组织培养基和粗组织提取物中发现的物质的非特异性干扰具有抗性。通过用环核苷酸磷酸二酯酶消化或用抗环磷酸腺苷抗体吸附去除可检测的环磷酸腺苷，与通过QAE - 葡聚糖凝胶色谱或依次在Dowex 50、氧化铝上纯化的组织提取物中的测量结果进行比较，以及通过稀释和回收率实验，确保了直接在粗组织提取物中测量环磷酸腺苷的可靠性。
