Stroop S D, Beavo J A
Department of Pharmacology, University of Washington, Seattle 98195.
J Biol Chem. 1991 Dec 15;266(35):23802-9.
Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)
对环磷酸鸟苷（cGMP）与天然的环磷酸鸟苷刺激的磷酸二酯酶以及两个缺乏催化结构域的独特分离胰凝乳蛋白酶片段结合的研究表明，该酶含有两个非催化性cGMP结合位点/同二聚体。在高浓度硫酸铵存在的情况下，每摩尔环磷酸鸟苷刺激的磷酸二酯酶同二聚体结合2摩尔cGMP。在这些条件下，可获得结合的线性Scatchard图，其表观解离常数（Kd）约为2微摩尔。加入3 - 异丁基 - 1 - 甲基黄嘌呤会产生曲线。在没有硫酸铵的情况下，cGMP从全酶上的解离很快，半衰期小于10秒，向孵育体系中加入硫酸铵会大大降低这种解离速率。在没有cGMP的情况下，天然酶对胰凝乳蛋白酶的降解具有抗性；然而，在其存在时，胰凝乳蛋白酶处理会产生几个离散的片段。同样，在有但不是没有cGMP的情况下，二环己基碳二亚胺会导致酶的不可逆激活，而不会使核苷酸与磷酸二酯酶交联。这两个观察结果都提供了证据，表明cGMP结合会导致酶的构象不同。只有cGMP结合时形成的构象容易受到胰凝乳蛋白酶的攻击或被二环己基碳二亚胺处理永久激活。cGMP结合暴露的一个主要胰凝乳蛋白酶切割位点在酪氨酸553处，这意味着该区域参与了构象变化。有限的蛋白水解实验表明，这些非催化结合位点位于先前提出的包含cGMP结合位点的内部序列同源区域内，并且它们对cGMP保持高亲和力和特异性，与酶的催化结构域无关。部分蛋白水解形成的产物可通过阴离子交换色谱法分离成单独的具有催化活性和cGMP结合活性的组分。对分离组分的凝胶过滤和电泳分析表明，cGMP结合峰具有二聚体结构。此外，它可以通过聚乙烯亚胺高效液相色谱法进一步分离为两个峰（峰IIIA和IIIB）。峰IIIA每摩尔二聚体结合2摩尔cGMP，表观Kd为0.2微摩尔。然而，峰IIIB的cGMP结合大大减少。用溴化氰对这些片段进行进一步消化表明，峰IIIA和IIIB之间的差异是由于IIIB中一个或多个额外的蛋白水解切口，这些切口去除了其C末端附近的一些残基，最可能是残基523 - 550或534 - 550。这反过来表明该区域对于cGMP结合活性至关重要。（摘要截断于400字）
