Rannels S R, Corbin J D
J Biol Chem. 1979 Sep 10;254(17):8605-10.
Monomeric cAMP-binding fragments of molecular mass 16,000 and 14,000 daltons were obtained by Sephadex G-75 chromatography of partially trypsin-hydrolyzed regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively. The Stokes radii were 19.1 and 16.4 A, the frictional ratios were 1.15 and 1.03, and the sedimentation coefficients were 1.94 and 1.91 S for the 16,000- and 14,000-dalton fragments, respectively. The 16,000-dalton fragment retained specific cyclic nucleotide binding characteristics of the native protein. The specificity of cyclic nucleotide binding to the 14,000-dalton fragment (cAMP greater than cIMP = 8-bromo-cAMP = 8-oxo-cAMP greater than cUMP = cGMP) differed from that of the native subunit (cAMP = 8-oxo-cAMP greater than 8-bromo-cAMP greater than cIMP greater than cUMP = cGMP). The 14,000-dalton fragment bound nearly 1 mol of cAMP/mol of fragment. The binding exchange rate of cAMP was much faster for the 14,000-dalton fragment than for either of the native regulatory subunits or for the 16,000 dalton fragment. Although hemin inhibited cAMP binding to the native regulatory subunits and to the 16,000 dalton fragment, the molecule did not affect cAMP binding to the 14,000-dalton fragment. Both of the native regulatory subunits and the isolated 16,000- and 14,000-dalton fragments could be covalently labeled with the photoaffinity analog, 8-N3-[32P]cAMP. The 14,000-dalton fragment could not be phosphorylated and neither fragment could recombine with the catalytic subunit to inhibit its activity. The results indicate that the functional entities of the regulatory subunit other than cAMP binding are destroyed by trypsin. The properties of the 16,000-dalton fragment suggest that the intact cAMP-binding site is contained in a small trypsin-resistant "core" of the native regulatory subunit. The properties of the 14,000-dalton fragment imply that part of the binding site of the native regulatory subunit was slighlty modified or lost during preparation of this fragment.
分别通过对环磷酸腺苷（cAMP）依赖性蛋白激酶同工酶I和II的部分胰蛋白酶水解调节亚基进行葡聚糖凝胶G - 75层析，获得了分子量为16,000道尔顿和14,000道尔顿的单体cAMP结合片段。对于16,000道尔顿和14,000道尔顿的片段，斯托克斯半径分别为19.1 Å和16.4 Å，摩擦比分别为1.15和1.03，沉降系数分别为1.94 S和1.91 S。16,000道尔顿的片段保留了天然蛋白的特异性环核苷酸结合特性。环核苷酸与14,000道尔顿片段的结合特异性（cAMP＞cIMP = 8 - 溴 - cAMP = 8 - 氧代 - cAMP＞cUMP = cGMP）与天然亚基的不同（cAMP = 8 - 氧代 - cAMP＞8 - 溴 - cAMP＞cIMP＞cUMP = cGMP）。14,000道尔顿的片段每摩尔片段结合近1摩尔的cAMP。14,000道尔顿片段的cAMP结合交换速率比天然调节亚基或16,000道尔顿片段都要快得多。虽然血红素抑制cAMP与天然调节亚基和16,000道尔顿片段的结合，但该分子不影响cAMP与14,000道尔顿片段的结合。天然调节亚基以及分离出的16,000道尔顿和14,000道尔顿片段都可以用光亲和类似物8 - N3 - [32P]cAMP进行共价标记。14,000道尔顿的片段不能被磷酸化，且两个片段都不能与催化亚基重新结合以抑制其活性。结果表明，调节亚基除cAMP结合之外的功能实体被胰蛋白酶破坏。16,000道尔顿片段的特性表明完整的cAMP结合位点包含在天然调节亚基的一个小的抗胰蛋白酶“核心”中。14,000道尔顿片段的特性意味着在该片段的制备过程中，天然调节亚基结合位点的一部分被轻微修饰或丢失。
