Interaction Of Phosphodiesterase Research Articles

Determinants for Phosphodiesterase 6 Inhibition by Its γ-Subunit

The interaction of phosphodiesterase 6 (PDE6) with its inhibitory Pgamma-subunits (Pgamma) is unparalleled among PDE families and is central to vertebrate phototransduction. The C-terminus of Pgamma occludes the active site of PDE6, thereby preventing hydrolysis of cGMP. In this study, we examine the determinants of this critical interaction using structure-based loss-of-function mutagenesis of a chimeric PDE5/PDE6 catalytic domain and gain-of-function mutagenesis of the PDE5 catalytic domain. This analysis revealed the key role of PDE6-specific residues within the catalytic domain M-loop-alpha-helix 15 region and suggested an important contribution of the H-loop-M-loop interface to the PDE6 inhibition by the Pgamma C-terminus. Identification of the determinants for the PDE6-Pgamma interaction offers insights into the evolution of the visual effector enzyme.

Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mitochondrial-bound hexokinase activity

The main objective of this study was to investigate the possible protective effect of pentoxifylline as a phosphodiesterase 5 inhibitor used as a cardiovascular medication on malathion-induced changes on rat mitochondrial-bound hexokinase activity. Animals in four various groups received moderate toxic dose of malathion (200 mg/kg/day), effective dose of pentoxifylline (50 mg/kg/day) alone and in combination, and the control group that received only vehicle. All administrations were done intraperitoneally for one week. At the end of the experiment, the brain was removed and the mitochondria were isolated. Hexokinase (HK) activity, cellular lipid peroxidation (LPO) and total antioxidant capacity (TAC) were analyzed in brain mitochondria. Malathion noticeably decreased TAC and increased HK activity and LPO in the mitochondria whereas pentoxifylline significantly restored malathion-induced changes in LPO, HK, and TAC. The results of the present study indicate that phosphodiesterase 5 inhibition remarkably protects brain mitochondria from malathion-induced changes on HK activity and oxidative stress.

A monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes.

The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.

Specific inhibition of cyclic AMP-dependent protein kinase, adenylate cyclase and phosphodiesterase by ATP and cyclic AMP analogs

The catalytic subunit of cyclic AMP-dependent protein kinase was isolated from pigeon breast muscle. The enzyme was 11,000-fold purified and, according to the data of SDS polyacrylamide gel electrophoresis, the enzyme preparation obtained was homogeneous. The molecular weight of the enzyme was estimated to be equal to 42,000 daltons. The pH optimum for the phosphotransferase reaction catalyzed by the catalytic subunit was 8.0. The kinetic mechanism of the reaction catalyzed by the cyclic AMP-dependent pigeon breast muscle protein kinase was studied. The results obtained permitted us to draw the conclusion that the reaction proceeded by the random Bi-Bi mechanism. Using the ATP analogs containing various reactive groups, a study was carried out on the ATP-binding site of the catalytic subunit. Among the ATP analogs tested, the compounds which irreversibly modified the active site of the enzyme were found. On the basis of the data on the kinetics of inhibition of the catalytic subunit by these analogs and pH dependence of the inactivation reaction, an assumption was made about possible involvement of the histidine residue in the catalytic action of the enzyme. A preparation of the solubilized rabbit heart adenylate cyclase possessing distinct catalytic activity was obtained. The effect of various ATP analogs on the solubilized adenylate cyclase was studied. Despite the presence of alkylating groups in different positions of the analogs tested, no covalent blocking of the active site of the enzyme was observed. All the compounds appeared to be competitive inhibitors. The affinity of the analogs with haloidalkyl groups in the phosphate chain increased with the increase in the number of phosphorus atoms. The 8-substituted ATP analogs that were likely to have syn-conformation possessed low affinity for the enzyme. The absence of a nucleophylic group in the active site of adenylate cyclase in the immediate proximity to the binding region of the γ-phosphorus atom of the ATP molecule was postulated. A partially purified phosphodiesterase preparation from rat liver, sufficiently specific towards cyclic AMP and characterized by two Michaelis constants for the cyclic nucleotide, namely 360 and 2.5–5 μ m, was obtained. The interaction of the phosphodiesterase with the synthesized cyclic AMP analogs containing reactive groups of various types in certain positions of the nucleotide molecule was studied. All compounds tested were found to be phosphodiesterase inhibitors acting on the active site of the enzyme; a number of these analogs provided irreversible inhibition. On the basis of the inhibitory analysis data, a tentative topography of the ATP-binding sites of the active centers of adenylate cyclase and protein kinase was proposed. Based on the results obtained in the study on the interaction of phosphodiesterase with cyclic AMP analogs, structural and functional characteristics of cyclic AMP-binding site of phosphodiesterase were determined.