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Phosphodiesterase (PDE) activity measured by micro‐isothermal calorimetry; a study of cyclic nucleotide analogues as substrates and inhibitors of PDEs1a, 1b, 1c, 2, 4, 5, 6, 8, 9, and 10
Author(s) -
Hinds Thomas Richard,
Poppe Heiko,
Tang XiaoBo,
Rybalkin Sergei D,
Butt Elke,
Beavo Joseph A
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.836.4
Subject(s) - nucleotide , cyclic nucleotide , chemistry , phosphodiesterase , kinetics , cyclic nucleotide phosphodiesterase , isothermal titration calorimetry , signal transduction , biophysics , biochemistry , enzyme , stereochemistry , biology , physics , quantum mechanics , gene
Identification of cellular signal transduction pathways that are hypothesized to be regulated by cyclic nucleotides often have been probed using various cyclic nucleotide analogues. However, the effect that these probes have on different PDEs has in general not been studied. Since radioactive analogs are not available, we employed micro isothermal calorimetry to measure the heat of hydrolysis of the analogs. Analogues that did not serve as substrates were tested to determine if they could be inhibitors. We show that this assay method gives similar results to the standard radioactive assay using tritiated cyclic nucleotides. Somewhat unexpectedly we find that many of the 11 most commonly used cyclic nucleotide analogues can act as substrates for multiple PDEs and most of the others are inhibitors. We also find that these analogues can inhibit different PDEs by various mechanisms including competitive, non‐competitive, and linear mixed kinetics. The kinetics obtained suggests that the active sites of dimeric PDEs must interact with one another in order to display non‐competitive and linear mixed kinetics. In addition, we provide evidence that inhibition of PDEs with so‐called selective analogs can indirectly increase both PKA and PKG mediated phosphoylation. Since these analogs can inhibit PDEs and thereby increase cAMP or cGMP, it is clear that care must be taken in the interpretation of results obtained when they are used for studying cellular regulatory pathways.