Identification, quantitation, and cellular localization of PDE1 calmodulin-stimulated cyclic nucleotide phosphodiesterases.
The calmodulin-stimulated cyclic nucleotide phosphodiesterases (PDE1s) constitute a large gene family and are found in a wide variety of tissues and cells. Because of the functional diversity of PDE1 genes and the observation that these isozymes often make up a major component of the total cyclic nucleotide hydrolytic activity in certain cell types, PDE1s are of growing interest as targets for therapeutic intervention. Here we describe a series of methodologies to identify, quantitate, and determine the cellular expression of PDE1 isozymes. We describe first the resolution of different PDEs using high-performance anion-exchange chromatography and then a Western blotting methodology for identifying or authenticating PDE1 activities. Next we present an immunoprecipitation method that can be used for quantitating specific PDE1 isoforms and describe the use of RNase protection analysis for further identification of PDE1 subtypes. Finally, we provide a simple, immunocytochemical method for determining the cellular expression of PDE1 isozymes. Combined, the above methodologies should allow an investigator to identify, quantitate, and determine the cellular localization of PDE1 isozymes in any tissue with little ambiguity.
Sonnenburg
W K
WK
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA. kajsa@u.washington.edu
Rybalkin
S D
SD
Bornfeldt
K E
KE
Kwak
K S
KS
Rybalkina
I G
IG
Beavo
J A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis* / Q000302:isolation & purification; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010215:Papio; D010727:Phosphoric Diester Hydrolases; D011233:Precipitin Tests; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism; D016415:Sequence Alignment
D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases