Abstract
Cyclic nucleotide phosphodiesterases (PDEs) are important in regulating cyclic nucleotide-mediated signal transduction pathways by catalyzing hydrolysis of cyclic nucleotides. In vascular smooth muscle cells, nitric oxide (NO) released from nerve endings stimulates a soluble guanylyl cyclase to increase cGMP production, and thus causes vascular smooth muscle relaxation and increases blood flow. On the other hand, PDE5 in smooth muscle cells hydrolyses cGMP and these muscle cells return to the more contracted state. VIAGRA? (sildenafil citrate) is efficacious in the treatment of male erectile dysfunction by inhibiting PDE5 function in smooth muscle cells.We performed polymerase chain reaction (PCR) to isolate the 2.7-kb cDNA encoding human PDE5A from human lung cDNA library. The cDNA was subcloned into pET-32a(+) vector and soluble recombinant human PDE5A was expressed in Escherichia coli BL21(DE3) with IPTG induction at 8℃. Besides, we developed a simple method based on HPLC for measuring the activity of PDE5A. The sensitivity of detection is as low as 1x10-11 mol of GMP. Using this method, the properties of recombinant human PDE5A purified by affinity column chromatography was characterized. The maximal enzyme activity was oberved at a temperature range of 37℃ to 40℃ and at a pH range of 8 to 8.5. The purified recombinant PDE5A was extremely labile. It was found to lose 65% of its original activity at 36℃ in 20 minutes. The PDE5A catalytic activity was increased by Mg2+, Co2+ or Mn2+, and inhibited by Zn2+, Ni2+ or Hg2+. Detergents, such as SDS and Triton, also inactivated PDE5A. EDTA at the concentration of 0.1 mM was stimulative to PDE5A catalytic activity, but inhibitory at 10 mM. Sildenafil also inhibited the recombinant PDE5A, and the IC50 value was found to be about 75 nM.