Gabibov A G, Kochetkov S N, Sashchenko L P, Severin E S
Biochim Biophys Acta. 1979 Aug 15;569(2):145-52. doi: 10.1016/0005-2744(79)90049-4.
The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of nonfluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180,000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions.
本文描述了一种用于测定猪脑中环磷酸腺苷（cAMP）及其类似物与环磷酸腺苷依赖性蛋白激酶结合的解离常数的方法。测量配体结合参数的技术基于乙烯基环磷酸腺苷（etheno cAMP）与蛋白激酶结合后荧光光谱的变化。通过这些类似物对乙烯基环磷酸腺苷的竞争性置换，测定了多种非荧光性环磷酸腺苷类似物的解离常数。发现猪脑蛋白激酶中环磷酸腺苷结合位点的数量为2.2；结合时未观察到协同性。研究中的蛋白激酶全酶复合物（Mr = 180,000）在天然条件下被确定为R2C2型化学计量比。
