Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Interaction of 125I-labeled Ca2+-dependent regulator protein with cyclic nucleotide phosphodiesterase and its inhibitory protein.

The Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase was labeled with 125I to the extent of 1 mol of monoiodotyrosine per mol. The iodinated protein showed a small decrease in affinity for phosphodiesterase but gave the same maximal level of activation of the enzyme as did the unmodified regulator protein. Iodinated regulator protein formed complexes with both highly purified cyclic nucleotide phosphodiesterase and phosphodiesterase inhibitory protein in the presence but not in the absence of Ca2+ as demonstrated by ultracentrifugation in glycerol gradients. Cross-linking experiments indicate that the Ca2+-dependent regulator protein interacts with the large subunit of the inhibitory protein.     

Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein.

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[125I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca2+-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by 125I-CDR was Ca2+-dependent and the labeled protein had a Ka for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. 125I-CDR formed a Ca2+-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autorradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.     

Inhibition of Ca2+-activated cyclic nucleotide phosphodiesterase reaction by a heat-stable inhibitor protein from bovine brain.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.     

Purification and Properties of a Unique Nucleotide Pyrophosphatase/Phosphodiesterase I That Accumulates in Soybean Leaves in Response to Fruit Removal

Several unique proteins accumulate in soybean (Glycine max) leaves when the developing fruits are removed. In the present study, elevated levels of nucleotide pyrophosphatase and phosphodiesterase I activities were present in leaves of defruited soybean plants. The soluble enzyme catalyzing these reactions was purified nearly 1000-fold, producing a preparation that contained a single 72-kD polypeptide. The molecular mass of the holoenzyme was approximately 560 kD, indicating that the native enzyme was likely octameric. The purified enzyme hydrolyzed nucleotide-sugars, nucleotide di- and triphosphates, thymidine monophosphate p-nitrophenol, and inorganic pyrophosphate but not nucleotide monophosphates, sugar mono- and bisphosphates, or NADH. The subunit and holoenzyme molecular masses and the preference for substrates distinguish the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I from other plant nucleotide pyrophosphatase/phosphodiesterase I enzymes. Also, the N-terminal sequence of the soybean leaf enzyme exhibited no similarity to the mammalian nucleotide pyrophosphatase/phosphodiesterase I, soybean vegetative storage proteins, or other entries in the data bank. Thus, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I appears to be a heretofore undescribed protein that is physically and enzymatically distinct from nucleotide pyrophosphatase/phosphodiesterase I from other sources, as well as from other phosphohydrolytic enzymes that accumulate in soybean leaves in response to fruit removal.     

Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain.

Abstract— Cyclic nucleotide phosphodiesterase activity (calcium-dependent and calcium-independent) and CDR (calcium-dependent regulator protein of phosphodiesterase) are present in all ten brain regions examined, with the specific activity of both being highest in areas predominating in grey matter and lowest in areas consisting largely of white matter. Fractionation of cerebral cortex into neuronal and glial perikarya shows that cyclic nucleotide phosphodiesterase and CDR are present at approximately equal levels in both cell types. Subcellular fractionation reveals that calcium-sensitive enzyme specific activity, as well as CDR are highly localized in the 100,000 g supernatant fraction. Regional, cellular and subcellular distribution studies indicate that the distribution of CDR closely parallels the distribution of enzyme specific activity, suggesting that the levels of cyclic nucleotide phosphodiesterase and CDR may be synchronized.     

Collagenase inhibition by cationic proteins derived from cartilage and aorta.

Modulator protein, a brain troponin C-like protein, has been coupled to Sepharose 4B using conditions that allow retention of phosphodiesterase stimulatory activity. This conjugate has been used to directly demonstrate the calcium dependent formation of a reversible modulator protein-phosphodiesterase complex and to purify a cyclic nucleotide phosphodiesterase by affinity chromatography.     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

CYCLIC NUCLEOTIDE METABOLISM IN ELECTROPLAX OF ELECTROPHORUS ELECTRICUS

Abstract— In order to describe the regulation of cyclic nucleotide metabolism in a cholinergic tissue, the properties of cyclic nucleotide phosphodiesterase were determined in electroplax of Electrophorus electricus and compared to those of mammalian brain. Electroplax phosphodiesterase was Mg²⁺ -dependent. localized in the soluble fraction and displayed normal linear Lineweaver-Burk kinetics ( K _m: cyclic AMP. 1.4 μ m ; cyclic GMP, 0.54 μ m ). No low affinity (i.e. high K _m) activity was detected. These results were correlated with comparatively low tissue levels of cyclic AMP (67 pmol/g) and cyclic GMP (3.2 pmol/g). Attempts were made to detect calcium-dependent phosphodiesterase because of the presence of large amounts of the calcium-dependent regulator protein (CDR) in electroplax, as this protein has been shown to activate brain phosphodiesterase. Assay with EGTA under a variety of conditions revealed that no calcium-dependent activity could be detected. Preparation of CDR-deficient phosphodiesterase also failed to produce calcium-dependent activity. Assay of phosphodiesterase in other cholinergic tissues revealed calcium-dependent activity in Electrophorus muscle and rat diaphragm but not in Torpedo electroplax. The results suggest that calcium-dependent activity is not a significant portion of phosphodiesterase in electroplax and indicate alternate roles for CDR in electric tissue.     

Cyclic GMP phosphodiesterase from bovine retina. Amino acid sequence of beta-subunit and nucleotide sequence of corresponding cDNA

Primary structure of beta-subunit of the cyclic GMP phosphodiesterase has been determined by the parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The beta-subunit contains 852 amino acid residues, its molecular mass is 98291 Da. A significant homology is found between beta- and alpha-subunit of the cGMP phosphodiesterase.     

Giardia lamblia: detection and characterization of calmodulin

Calmodulin was detected in Giardia lamblia by radioimmunoassay and cyclic AMP phosphodiesterase activation. This protein was purified to apparent homogeneity by fast protein liquid chromatography with a yield of 260 ng of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis, gel filtration, and ion exchange chromatography. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes, e.g., an apparent molecular weight of 16.7 kD; activation in calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase; and sensitivity to known calmodulin antagonists.     

Modified cyclic nucleotide systems in Morris hepatoma 3924A favoring expression of cyclic GMP effect.

Modifications in the cyclic nucleotide systems favoring the expression of cyclic GMP effects were found to occur in the transplanted fast-growing Morris hepatoma 3924A. These included: (a) a decreased level of cyclic GMP phosphodiesterase and an increased level of cyclic AMP phosphodiesterase; (b) a disproportionately increased level of cylic GMP-dependent protein kinase relative to that of cyclic AMP-dependent protein kinase; (c) a disproportionately increased level of stimulatory modulator of cyclic AMP-dependent protein kinase relative to that of inhibitory modulator of cyclic AMP-dependent protein kinase; and (d) an increased level of phosphoprotein phosphatase.     

Regulation of the high Km cyclic nucleotide phosphodiesterase of adrenal medulla by the endogenous calcium-dependent-protein activator.

A cyclic nucleotide phosphodiesterase sensitive to the calcium-dependent endogenous protein activator has been identified in rat and beef adrenal medulla. In this tissue the ratio between the activity of this enzyme and that of the low Km enzyme is smaller than the corresponding ratio in rat brain. The activator-sensitive phosphodiesterase, isolated from beef adrenal medulla has a high Km for cyclic 3,5-AMP. Saturating concentrations of the calcium dependent protein activator decreased significantly the apparent Km of this enzyme using cAMP as a substrate.     

Modified cyclic nucleotide systems in Morris hepatoma 3924A favoring expression of cyclic GMP effect

Modifications in the cyclic nucleotide systems favoring the expression of cyclic GMP effects were found to occur in the transplanted fast-growing Morris hepatoma 3924A. These included: (a) a decreased level of cyclic GMP phosphodiesterase and an increased level of cyclic AMP phosphodiesterase; (b) a disproportionately increased level of cyclic GMP-dependent protein kinase relative to that of cyclic AMP-dependent protein kinase; (c) a disproportionately increased level of stimulatory modulator of cyclic GMP-dependent protein kinase relative to that of inhibitory modulator of cyclic AMP-dependent protein kinase; and (d) an increased level of phosphoprotein phosphatase.     

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

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodiesterase 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.     

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

Some biochemical properties of alkyl phosphotriesters of cyclic AMP.

The biochemical properties of several alkyl phosphotriesters of cyclic AMP were studied with respect to their interactions with beef heart protein kinase and cyclic nucleotide phosphodiesterase. Ethyl and propyl triesters did not enhance the phosphorylation of histone by protein kinase and methyl, ethyl, propyl and butyl triesters were poor competitors for the cyclic AMP binding site of the enzyme. However, these alkyl phosphotriesters were effective inhibitors of cyclic nucleotide phosphodiesterase with the K$\text{i}$'s arrayed in the following order: methyl > ethyl > propyl > butyl > cetyl triester. Metabolic studies with mice indicated that intraperitoneal injection of low doses of propyl triester for one week significantly increased cyclic AMP concentration.     

Serum modification of cyclic nucleotide phosphodiesterase forms independent of protein synthesis.

Addition of 10% fetal calf serum to BHK cells made quiescent by maintenance for 48 hours in sub-optimal serum (0.5%) caused rapid changes in cyclic AMP phosphodiesterase activity (increased maximum velocity and affinity) even in the presence of inhibitors of protein synthesis. Activity changes were associated with an alteration in the number of forms of cyclic AMP phosphodiesterase identified by Agarose gel filtration. Three forms of cyclic nucleotide phosphodiesterase were apparent after serum addition whereas only two forms were resolved in quiescent BHK cells. The initial rapid increase in cyclic AMP phosphodiesterase activity seen when serum was added to quiescent cells was followed temporally by a much slower increase in cyclic AMP phosphodiesterase activity that could be prevented by cycloheximide or actinomycin D.