title: Cloning of dog heart PDE1A - a first detailed characterization at the molecular level in this species.
abstract: The dog, as a model for cardiovascular function, has been widely used in the pharmacological analysis of PDE inhibitors, particularly those thought to target the heart. However biochemical analyses of dog heart PDE have been largely performed on mixed enzyme populations, sequence information is lacking and no PDE from dog heart has been cloned. We have characterized a completely purified PDE1 enzyme from dog heart using dye-affinity, Mono-Q and calmodulin-affinity chromatography. The enzyme was stimulated 3-4-fold by calmodulin ([S]=0.5 microM) and, in the absence of calmodulin, exhibited biphasic kinetics with a low K(m) of 1.2 microM and 0.53 microM for cAMP and cGMP, with respective V(max) values of 283 and 146 nmoles min(-1) mg(-1). Internal peptides from this enzyme were used to design degenerate PCR primers. Subsequent 3'-RACE, 5'-RACE and high fidelity PCR were then used to produce a full length gene identified as PDE1A1 by sequence identity to human and bovine sequences. Northern analysis using the dog heart cDNA as a probe suggested the presence of an additional form of PDE1, in heart only, separate from the PDE1A group which was present in both heart and skeletal muscle. Multiple forms of human PDE1A are known to exist and PDE1B is present in human heart muscle. The findings here extend the PDE1 data to the dog and contribute to our understanding of the molecular biology of PDE1A in this species.
authors.affiliation: Department of Crystallography, Birkbeck College, Malet Street, WC1E 7HX, London, UK.
authors.forename: J C
authors.initials: JC
authors.lastname: Clapham
authors.forename: A F
authors.initials: AF
authors.lastname: Wilderspin
mesh_terms: D000595:Amino Acid Sequence; D000676:Amrinone / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004285:Dogs / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
chemical_list: D002147:Calmodulin; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; C517265:PDE1A protein, human; C517266:PDE1B protein, human; D000676:Amrinone