Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, United States.
Cell Signal. 2011 Oct;23(10):1578-83. doi: 10.1016/j.cellsig.2011.05.005. Epub 2011 May 17.
Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30°C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn(++) or Mg(++) caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.
磷酸二酯酶-5（PDE5）是一种二聚体，每个亚基包含一个 cGMP 特异性催化结构域和一个变构 cGMP 结合亚基（GAF A）。PDE5 表现出三种构象形式，可以通过 NativePAGE 分离，按迁移率递减的顺序表示为 Band 1、2 和 3。一种主要由 Band 2 PDE5 组成的制剂，通过与 cGMP 或 PDE5 特异性抑制剂西地那非、伐地那非或他达拉非孵育 1 小时，部分转化为 Band 3 PDE5，但不能与 cAMP、米力农（PDE3 特异性）或 rolipram（PDE4 特异性）孵育。Band 2 PDE5 在 30°C 下与西地那非孵育过夜几乎完全转化为 Band 3 PDE5。这种时间依赖性转化伴随着变构 cGMP 结合活性增加 7 倍，表明 Band 3 PDE5 比 Band 2 PDE5 更适合变构 cGMP 结合。通过与 cGMP 预孵育（cGMP 结合 PDE5 的变构和催化位点），Band 2 PDE5 向 Band 3 PDE5 的转化速度比与催化位点特异性西地那非的转化速度更快。Band 2/Band 3 PDE5 混合物与 EDTA 孵育过夜会导致时间依赖性转化为 Band 1 PDE5（脱辅基酶），并且这种转化伴随着 cGMP 结合活性丧失 50%。用 EDTA 孵育后，加入 Mn(++) 或 Mg(++) 会导致 Band 1 恢复为 Band 2/Band 3 PDE5 混合物，其中 Band 3 PDE5 占优势。这种恢复伴随着变构 cGMP 结合活性增加 3 倍。结果表明，生理条件下 cGMP 和/或二价金属离子占据催化结构域会导致 Band 2 向 Band 3 PDE5 的转化，从而增加酶的变构 cGMP 结合。这种转化会通过增加 PDE5 变构 cGMP 结合位点的 cGMP 隔离和增加酶的催化位点的 cGMP 降解来增加对 cGMP 作用的负反馈效应。这种转化还会增加 PDE5 抑制剂与酶的结合。
