Bohnert J L, Malencik D A, Anderson S R, Teller D, Fischer E H
Biochemistry. 1982 Oct 26;21(22):5563-70. doi: 10.1021/bi00265a028.
Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.
使用荧光腺苷环3',5'-磷酸（cAMP）类似物1,N6-乙烯腺苷环3',5'-磷酸（ε-cAMP）进行的荧光强度和各向异性测量对ε-cAMP的解离敏感，当cAMP依赖性蛋白激酶的I型或II型调节亚基（RI或RII）与催化亚基结合时，ε-cAMP就会发生解离。使用ε-cAMP的研究表明，MgATP对两种类型蛋白激酶的重组有相反的影响：MgATP强烈稳定I型全酶，而它略微使II型全酶不稳定。当以10 microM浓度测试时，合成底物肯普肽对两种全酶的重组有轻微的抑制作用。蛋白激酶抑制剂有更大的作用，这在I型酶的重新结合中尤为明显。I型调节亚基与蛋白激酶抑制剂竞争的相对能力降低，表明两种相反平衡（ε-cAMP和催化亚基结合）的综合作用对于两种类型的调节亚基是不同的。置换实验表明，cAMP和ε-cAMP与I型亚基的结合能力大致相同。两种调节亚基与ε-cAMP结合时伴随的缓慢构象变化在全酶存在时大大加速，这表明全酶的解离是通过三元复合物发生的。ε-cAMP结合的时间进程也显示了RII结合特性的异质性。II型亚基的37000道尔顿片段保留了天然亚基的ε-cAMP结合特性。然而，只有一部分片段制剂（根据沉降测量估计约为32%）能很好地结合催化亚基，这表明切割存在异质性。
