Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase.
Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase.
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DOI：
10.1021/bi00265a028
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发表时间：
1982-10
期刊：
影响因子：
2.9
通讯作者：
J. L. Bohnert;D. Malencik;S. Anderson;D. Teller;E. Fischer
中科院分区：
文献类型：
--
作者：
J. L. Bohnert;D. Malencik;S. Anderson;D. Teller;E. Fischer
使用荧光环3′，5′-磷酸腺苷（cAMP）类似物1，n6 -乙烯腺苷环3′，5′-磷酸（epsilon-cAMP）测量的荧光强度和各向异性对epsilon-cAMP的解离很敏感，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，发生这种解离。使用epsilon-cAMP的研究表明，MgATP对两种类型的蛋白激酶的重构具有相反的作用：MgATP对I型全酶具有很强的稳定性，而对II型全酶具有轻微的不稳定性。合成的底物Kemptide在10 μ m浓度下对两种全酶的重组均有较小的抑制作用。蛋白激酶抑制剂的作用更大，在I型酶的再结合中尤为明显。I型调控亚基与蛋白激酶抑制剂竞争的相对能力减弱表明，两种对立平衡（epsilon-cAMP和催化亚基结合）对两种类型的调控亚基的综合作用是不同的。位移实验表明cAMP和epsilon-cAMP与I型亚基的结合效果差不多。两种调节亚基结合epsilon-cAMP的缓慢构象变化随着全酶的加入而大大加速，这表明全酶的解离是通过三元配合物发生的。epsilon-cAMP结合的时间过程也显示了RII结合特性的异质性。II型亚基的37000道尔顿片段保留了原生亚基的epsilon-cAMP结合特性。然而，只有一小部分制备的碎片（根据沉降测量估计约32%）能很好地结合催化亚基，这表明裂解的异质性。
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.