Lin Guiting, Xin Zhong-Cheng, Lue Tom F, Lin Ching-Shwun
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California-San Francisco, CA 94143, USA.
J Urol. 2006 Sep;176(3):1242-7. doi: 10.1016/j.juro.2006.04.031.
We determined the differential cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity of phosphodiesterase-5 isoforms and the inhibitory effects of sildenafil (Pfizer, New York, New York) and vardenafil (Bayer Pharmaceutical Research, Wuppertal, Germany).
Coding sequences of the human phosphodiesterase-5 isoforms A1, A2 and A3 were cloned into pBlueBac4.5/V5-His (Invitrogen, Carlsbad, California), which allows the tagging of histidines at the carboxyl terminal of the expressed protein. Isoforms were expressed with the Bac-N-Blue baculoviral system and purified with the ProBond system. Expression clones were identified by polymerase chain reaction using vector and phosphodiesterase-5 specific primers. Purified proteins were verified by Western blotting. Purified proteins were analyzed for cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity, and used to determine the differential potencies of the phosphodiesterase-5 selective inhibitors sildenafil and vardenafil.
Cloning and expression of phosphodiesterase-5A1 to A3 isoforms in the baculoviral system resulted in the isolation of purified isoform proteins. Mean cyclic guanyl monophosphate catalytic activity (K(m)) +/- SD was 4.76 +/- 0.37, 4.52 +/- 0.09 and 11.39 +/- 0.22 microM for A1 to A3, respectively. Mean cyclic guanyl monophosphate binding activity (K(d)) was 3.24 +/- 0.47, 1.95 +/- 0.60 and 1.70 +/- 0.47 microM for A1 to A3, respectively. Mean IC(50) of sildenafil against phosphodiesterase-5A1 to A3 was 1.20 +/- 0.34, 2.83 +/- 0.56 and 2.28 +/- 0.38 nM, respectively. Mean IC(50) of vardenafil against phosphodiesterase-5A1 to A3 was 0.41 +/- 0.15, 0.23 +/- 0.08 and 0.45 +/- 0.06 nM, respectively.
Phosphodiesterase-5A1 and A2 had similar K(m) values, while phosphodiesterase-5A3 had a much higher K(m) and, thus, lower cyclic guanyl monophosphate catalytic activity. Phosphodiesterase-5A2 and A3 had similar K(d) values, while phosphodiesterase-5A1 had higher K(d) and, thus, lower cyclic guanyl monophosphate binding activity. Vardenafil was more potent (3 to 12-fold) than sildenafil for inhibiting the catalytic activity of all 3 human phosphodiesterase-5 isoforms with phosphodiesterase-5A2 showing the highest differentiation (12-fold).
我们测定了磷酸二酯酶-5亚型的环磷酸鸟苷催化活性和环磷酸鸟苷结合活性差异，以及西地那非（辉瑞公司，纽约，纽约州）和伐地那非（拜耳制药研究公司，德国伍珀塔尔）的抑制作用。
将人磷酸二酯酶-5亚型A1、A2和A3的编码序列克隆到pBlueBac4.5/V5-His（英杰公司，加利福尼亚州卡尔斯巴德）中，该载体可在表达蛋白的羧基末端标记组氨酸。使用Bac-N-Blue杆状病毒系统表达亚型，并使用ProBond系统进行纯化。使用载体和磷酸二酯酶-5特异性引物通过聚合酶链反应鉴定表达克隆。通过蛋白质印迹法验证纯化的蛋白质。分析纯化的蛋白质的环磷酸鸟苷催化活性和环磷酸鸟苷结合活性，并用于测定磷酸二酯酶-5选择性抑制剂西地那非和伐地那非的差异效价。
在杆状病毒系统中克隆并表达磷酸二酯酶-5A1至A3亚型，得到纯化的亚型蛋白。A1至A3的平均环磷酸鸟苷催化活性（K(m)）±标准差分别为4.76±0.37、4.52±0.09和11.39±0.22 microM。A1至A3的平均环磷酸鸟苷结合活性（K(d)）分别为3.24±0.47、1.95±0.60和1.70±0.47 microM。西地那非对磷酸二酯酶-5A1至A3的平均IC(50)分别为1.20±0.34、2.83±0.56和2.28±0.38 nM。伐地那非对磷酸二酯酶-5A1至A3的平均IC(50)分别为0.41±0.15、0.23±0.08和0.45±0.06 nM。
磷酸二酯酶-5A1和A2具有相似的K(m)值，而磷酸二酯酶-5A3具有更高的K(m)，因此环磷酸鸟苷催化活性更低。磷酸二酯酶-5A2和A3具有相似的K(d)值，而磷酸二酯酶-5A1具有更高的K(d)，因此环磷酸鸟苷结合活性更低。在抑制所有3种人磷酸二酯酶-5亚型的催化活性方面，伐地那非比西地那非更有效（3至12倍），其中磷酸二酯酶-5A2表现出最高的差异（12倍）。
