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3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5—being a regulator of vascular smooth muscle contraction—is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors. [ABSTRACT FROM AUTHOR]

The kinetic behavior of cGMP-specific phosphodiesterase in a totally bleached bovine retinal rod outer segment suspension was studied by the pH-metric method at high and low concentrations of free calcium ions (≈100 μM and <10 nM, respectively). The phosphodiesterase was activated by low GTP concentrations (∼1-2 μM) that were comparable with the concentration of G-protein transducin, the GTP-binding alpha-subunit of which is the intrinsic activator of photoreceptor phosphodiesterase. The results allow the suggestion that, besides the earlier described system of RGS proteins participating in acceleration of GTP hydrolysis, rod outer segments also contain an additional Ca-dependent mechanism to inactivate so-called 'free' transducin, i.e. active transducin that has not managed to interact with phosphodiesterase within the time restricted by the duration of photoreceptor response. [ABSTRACT FROM AUTHOR]

The kinetic behavior of phosphodiesterase activated by transducin in a complex with a non-hydrolyzable GTP analogue (guanosine-5′-O-thiotriphosphate) was studied by the pH-metric method in preparations of light-adapted external segments of bovine retinal rods in a range of magnesium ion concentrations from 0.4 to 20 mM. These results indicate that when using the reaction media containing 10-15 mM Mg ions, introduction of 2-4 mM ethylene glycol tetraacetic acid (a chelator of calcium ions) in the reaction medium induces only relatively small changes in the concentration of free magnesium ions that are not able to significantly influence the phosphodiesterase activity. [ABSTRACT FROM AUTHOR]