Assays for cyclic nucleotide-specific phosphodiesterases (PDEs) in the central nervous system (PDE1, PDE2, PDE4, and PDE10).
Since the identification of phosphodiesterase activity in brain tissue more than 40 years ago, 11 distinct gene families have been identified, differing with respect to localization, regulation, affinity for cAMP and cGMP, and distinct functions within cells. PDEs 1, 2, 4, and 10 are currently of special interest to CNS pharmacology because of their high expression in specific areas of the brain and the behavioral effects of inhibitors of these enzymes in rodents. Efficient high-throughput PDE enzyme assays are essential for PDE-targeted drug discovery, and this unit details two types of assays. The first method is relatively inexpensive and is based on separating radiolabeled cNMPs from degradation products on alumina columns. The second method is fluorescence-based; it is fast and better accommodates high-throughput screening, but is more expensive. Although these methods have successfully been used for PDEs 1, 2, 4 and 10, they could be readily adapted to other PDEs.
Deng
Chengjun
C
Memory Pharmaceuticals Corp, Montvale, New Jersey, USA.
Wang
Daguang
D
Bugaj-Gaweda
Bozena
B
De Vivo
Michael
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002490:Central Nervous System / Q000201:enzymology*; D002845:Chromatography / Q000379:methods*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000302:isolation & purification / Q000378:metabolism; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000302:isolation & purification / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000302:isolation & purification / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000302:isolation & purification / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism
D004800:Enzymes, Immobilized; C119843:PDE10A protein, human; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4