Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
Cell Signal. 2011 May;23(5):778-84. doi: 10.1016/j.cellsig.2010.12.005. Epub 2010 Dec 25.
Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg(++) or Mn(++), which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me(++)) with affinity of ~0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn(++). The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me(++); however, the cGMP- or sildenafil-induced shift was inhibited and the Me(++)-induced shift was facilitated by treatment with EDTA. Results suggested that Me(++) removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me(++), substrate, or substrate analog. It is concluded that Me(++) causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.
经天然聚丙烯酰胺凝胶电泳（native PAGE）显示，纯化的 PDE5 制剂表现出两种迁移形式（带 2 和带 3）的可变比例。用 cGMP 或底物类似物（如西地那非）处理重组或天然 PDE5，已知这两种物质都能对酶功能产生刺激作用，导致类似的天然 PAGE 带向低迁移形式（带 2 向带 3 的迁移）转移。用 Mg(++)或 Mn(++)孵育 PDE5，已知这两种物质能刺激活性，导致酶从带 2 向带 3 的类似转移，与 cGMP 或西地那非一样，但用 EDTA 孵育会导致时间和浓度依赖性的向高迁移（带 2 和带 3 向带 1 的迁移）。EDTA 诱导的带转移的缓慢时程表明，一种预先结合的金属离子（Me(++))被去除，其亲和力约为 0.1 nM，与之前确定的 PDE5 对 Zn(++)的亲和力相似。EDTA 处理的酶（带 1）可以通过添加 cGMP、西地那非或 Me(++)转移到带 2 和带 3；然而，cGMP 或西地那非诱导的转移被抑制，Me(++)诱导的转移被 EDTA 处理促进。结果表明，从 PDE5 中去除 Me(++)会产生一种独特的 apoenzyme 形式（带 1，更球形，带负电荷，或两者兼有），这种 apoenzyme 形式可以通过添加 Me(++)、底物或底物类似物部分转化为形式（带 2，带负电荷或带负电荷较少，或两者兼有；带 3，更细长/带正电荷，或两者兼有）。结论是，Me(++)导致 PDE5 转化为与底物或抑制剂结合到催化位点所引起的类似构象形式。
