Owens J R, Haley B E
J Supramol Struct. 1978;9(1):57-68. doi: 10.1002/jss.400090107.
Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3',5'-cyclic monophosphate (8-N3 cAMP) and [gamma32P]ATP, the membrane-binding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [32P]8-N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [gamma32P]ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg.ATP concentration is below approximately 10 micrometer. The catalytic subunit is not required for the Mg.ATP inhibition of 8-N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied on other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg.ATP.
利用放射性标记的环磷酸腺苷（cAMP）光亲和类似物8-叠氮腺苷-3',5'-环一磷酸（8-N3 cAMP）和[γ32P]ATP，对人红细胞膜cAMP激活的蛋白激酶的调节亚基和催化亚基的膜结合特性进行了研究。[32P]8-N3 cAMP用于定位和定量调节亚基。利用[γ32P]ATP使特定膜蛋白磷酸化增加来确定催化亚基的存在。数据支持一种机制，该机制通过紧密结合在膜上的调节亚基和催化亚基起作用，当存在cAMP且Mg.ATP浓度低于约10微摩尔时，催化亚基从膜上释放。Mg.ATP对8-N3 cAMP结合的抑制作用不需要催化亚基。用ATP的光亲和类似物8-叠氮三磷酸腺苷（8-N3ATP）进行的实验支持了ATP水解和磷酸化不参与调节的假说。数据表明调节亚基含有一个ATP调节位点，该位点抑制8-N3 cAMP结合和催化亚基的释放。这些结果表明，膜结合的I型酶（IM型）与在其他组织中研究的可溶性酶（IS型）有显著差异。这些酶通过处于不同的细胞位置而被分隔开，并且受到Mg.ATP的不同调节。
