磷酸二酯酶 cyclic nucleotide phosphodiesterases (PDEs) 是由一群酵素所組成的superfamily，可以降解細胞內的二級訊息傳遞物 cyclic AMP(cAMP) 及cyclic GMP(cGMP) 。其中專一性的cGMP-binding cGMP-specific phosphodiesterase (PDE5) 存在於許多組織細胞中，可以降解cGMP並且調節細胞內cGMP的含量，其中也包括了陰莖海綿體的平滑肌細胞。著名的威而剛 (Sildenafil; Viagra)與 犀利士(Tadalafil; Cialis)是兩個主要的PDE5抑制劑，藉由抑制PDE5的活性使cGMP在海綿體的平滑肌細胞內堆積，促使平滑肌的放鬆造成充血及勃起。
本實驗是將人類PDE5基因，包含N端融合蛋白質Thioredoxin整段轉殖到一冷休克表現質體，即為pCold-PDE5A。此表現質體經由轉型作用到E.coli strain BL21(DE3)中，並且在15℃下加入1mM IPTG後誘導24小時可得到大量表現的可溶性蛋白質。接著利用Ni2+-IDA 親和性管柱做蛋白質純化。而存在於不可溶包涵體的蛋白質則是利用on-column chemical refolding方法純化。本實驗利用一種簡單的reverse-phase HPLC儀器測定人類重組PDE5活性。利用此方法可以在15分鐘內測定受質 (cGMP)和反應產物(GMP)，其偵測反應產物GMP的靈敏度約在2x10-11 moles。經由實驗結果發現純化後的人類重組PDE5蛋白質其Km值在1.12- 1.27uM。Tadalafil (Cialis)可以有效的抑制人類重組PDE5蛋白質的活性，其IC50 約為 39nM，而Ki値約為3.7 nM。但是由不可溶包涵體 (inclusion bodies)所純化出來的重組蛋白質則是偵測不到酵素活性。本實驗也利用HPLC建立了一個可以偵測兩種PDE5抑制劑 Sildenafil 和Tadalafil的方法。分析五種商業藥品對於PDE5的活性發現皆具有很強的抑制作用，經由HPLC分析也證實了其中包含了Sildenafil 或Tadalafil兩種PDE5抑制劑之一。

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular secondary messengers cyclic AMP (cAMP) and cyclic GMP (cGMP). The cGMP-binding cGMP-specific phosphodiesterase (PDE5) degrades cGMP and regulates the intracellular level of cGMP in many tissues including the smooth muscle of the corpus cavernosum. Sildenafi (Viagra) and Tadalafil (Cialis) are two specific PDE5 inhibitors, promote penile erection by blocking PDE5 activity, which causes cGMP accumulation in the corpus cavernosum and trigger smooth muscle relaxation and increases blood flow.
In this study, a cDNA of thioredoxin- human PDE5 fusion protein was inserted into a cold-shock expression vector, pCold-PDE5A. This plasmid was transformed into E.coli strain BL21(DE3) and the recombinant protein was induced with 1mM IPTG at 15℃ for 24 hours. The soluble fraction of the fusion protein was purified by Ni2+-IDA affinity column. A simple method based on reverse-phase HPLC has been development for measuring the activity of recombinant human PDE5 protein. It allows quantization of product (GMP) and substrate (cGMP) in less than 15 min, and the sensitivity of detection is as low as 2x10-11 moles of GMP. The Km value of soluble human recombinant PDE5 protein is 1.12- 1.27 uM. Tadalafil (Cialis) potently inhibited the recombinant PDE5 protein with IC50 values of 39 nM, and the Ki value is 3.7 nM. The protein from inclusion bodies was also purified by on-column chemical refolding processes. However, the activity of recombinant Trx-PDE5 protein from inclusion bodies was not detected. We also established a method based on HPLC to detect two PDE5 inhibitors, Sildenafil and Tadalafil. Five different commercial drugs shown great inhibition of PDE5 activity were subjected to HPLC analysis, and it revealed that all of them contain either Sildenafil or Tadalafil.

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