Cytochemical localization of adenylate cyclase and 3',5'-nucleotide phosphodiesterase in Dictyostelium.

Cytochemical localizations of adenylate cyclase and 3′,5′-nucleotide phosphodiesterase were performed on aggregating Dictytostelium discoideum myxamoebae. The adenylate cyclase reaction product was localized on the inner surface of the plasma membrane. The phosphodiesterase reaction product was localized on the outer surface of the plasma membrane. Differences in enzyme activity were noted according to the state of cell (isolated or aggregated) and according to the cell position in larger aggregates. Heavy precipitation indicative of adenylate cyclase activity was not observed in isolated amoebae, but was often observed in streams and in some cells of aggregates. The precipitation indicative of phosphodiesterase activity could be found in isolated amoebae and in peripheral cells of aggregates.     

Cyclic 3':5'-nucleotide phosphodiesterase determined in various human tissues by DEAE-cellulose chromatography.

Tissue extracts from human heart, lung, liver, kidney, skeletal muscle and cerebrum displayed at least 3 distinct cyclic 3':5'-nucleotide phosphodieterase (EC 3.1.4.17) activity peaks (FI, FII, FIII) on DEAE-cellulose chromatography and various properties of these forms were compared in each tissue. FI eluted at about 0.08 M sodium acetate, hydrolyzed cyclic GMP more rapidly than it did cyclic AMP, and cyclic GMP hydrolysis by FI in most tissues was enhanced by a protein activator in the presence of CaCl2. As only high concentrations of cyclic AMP inhibited cyclic GMP hydrolytic activity of FI, the enzyme probably has a low affinity for cyclic AMP. FII eluted at about 0.2 M sodium acetate, hydrolyzed both nucleotides at equal rates, and substrate affinities were relatively low. Cyclic GMP hydrolysis by FII was also stimulated by addition of a protein activator in the presence of CaCl2 and cyclic AMP hydrolysis in this fraction was accelerated by a micromolar fraction of cyclic GMP. FII eluted at about 0.35 M hydrolyzed cyclic AMP preferentially and was insensitive to protein activator. These two cyclic nucleotides act as mutual inhibitors of the hydrolysis in this fraction. Ratio of the cyclic GMP to cyclic AMP hydrolysis was in the order FI, FII, FIII. Four activity peaks were eluted from the cerebral extract and enzymes from this tissue exhibited much the same properties as observed in the other tissues examined herein.     

Cyclic 3':5'-nucleotide phosphodiesterase. Stimulation of bovine brain cytoplasmic enzyme by lysophosphatidylcholine.

Brain cytoplasmic cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) requires an endogenous Ca2+-binding protein for ful activity. We now show that lysophosphatidylcholine also effectively enhances activator-deficient phosphodiesterase activity. Stimulation by both ligands was immediate and reversible; both rendered the enzyme more thermally labile, decreased the energy of activation, and increased the Vmax of phosphodiesterase without affecting its apparent Km for adenosine 3'5'-monophosphate. However, the cofactor requirements of the two ligands were different. Although the protein activator gave a greater stimulation than lysophosphatidylcholine, the simultaneous presence of the two gave a stimulation comparable to lysophosphatidylcholine, suggesting that the effect of the latter was predominant. Phosphodiesterase was also stimulated by oleic acid, cardiolipin, and phosphatidylinositol, albeit to a less extent.     

A new histo- and cytochemical method for demonstration of cyclic 3',5'-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

Cyclic 3',5'-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo- and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5' nucleotidase activity instead of an exogenous 5' nucleotidase source (snake venom). Cyclic GMP and was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5' nucleotidase was also studied utilizing 5' AMP or 5' GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5' nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

A heat-stable protein inhibitor of cyclic 3',5'-nucleotide phosphodiesterase.

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodieterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

Cyclic 3'.5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP

The rate of cyclic AMP hydrolysis by a cyclic 3′,5′-nucleotide phosphodiesterase was diminished by the presence of a cyclic AMP binding protein in the reaction mixture. The reduction was proportional to the concentration of the binding protein; and was more pronounced at 0° than at 30°, presumably because the affinity of cyclic AMP to the binding protein was greater at 0° (“apparent dissociation constant” = 3 × 10⁻⁸ M) than at 30° (“apparent dissociation constant” = 4 × 10⁻⁷ M). These experiments indicate that cyclic AMP bound to the binding protein is not susceptible to the action of phosphodiesterase. It is hydrolyzed only when dissociated from the protein, and the rate of dissociation appears to be the limiting factor. The possible physiological significance of these results is discussed.