Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain.
We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.
Giordano
D
D
Dipartimento do Biologia Cellulare e dello Sviluppo, Università di Roma La Sapienza, Rome, Italy.
De Stefano
M E
ME
Citro
G
G
Modica
A
A
Giorgi
M
M
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D051379:Mice; D009206:Myocardium / Q000201:enzymology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011233:Precipitin Tests; D011687:Purines; D011688:Purinones / Q000494:pharmacology; D000068677:Sildenafil Citrate; D013450:Sulfones; D014020:Tissue Extracts / Q000737:chemistry; D014407:Tumor Cells, Cultured
D007106:Immune Sera; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D011688:Purinones; D013450:Sulfones; D014020:Tissue Extracts; D000068677:Sildenafil Citrate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517382:Pde5a protein, mouse; C011145:zaprinast