Specific Phosphodiesterase Activity Research Articles

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Deletion and Characterization of Two Signaling Genes in Streptomyces

Streptomyces coelicolor is a filamentous, high G‐C (guanine‐cytosine), gram‐positive bacterium that produces an aerial mycelium. The genome consists of a 8,667,507 base pair linear chromosome for this organism and contains the largest number of genes so far discovered in a bacterium. It belongs to the phylum Actinobacteria. In addition, streptomycetes are used to produce the majority of antibiotics in the medical field. This study focuses on the effect of the deletion of Sco5218, which encodes a putative integral membrane protein and Sco5219, which encodes a putative lipoprotein in Streptomyces coelicolor. Both of these genes are found in a potential operon. The goal of this research is to construct the double mutant and discern differences in phenotype compared to the single deletion mutants of each gene. We hypothesize that the double mutant will have an enhanced effect on the sporulation defect of Streptomyces. Sco5218 is believed to contain an HD‐GYP domain through bioinformatics analysis and is highly conserved throughout the Streptomyces genus. The HD‐GYP domain is typically associated with cyclic di‐GMP specific phosphodiesterase activity. Following REDIRECT protocol, the Streptomyces coelicolor cosmid clone was introduced into E. coli via electroporation. PCR was then utilized for the deletion of Sco5218 and Sco5219. This PCR product was run onto a gel and purified. This pure PCR product was then transformed into the plasmid vector which will then be mated into Streptomyces coelicolor. The relevance of this research allows us to see the importance of the HD‐GYP domain in bacteria. We witnessed a slight delay in sporulation with the Sco5218 mutant deletion. With the added deletion of Sco5219 we hypothesize that there will be an enhanced delay in sporulation. If we witness a delay in the sporulation with the double mutant in Streptomyces we can deduce that both genes play a role in bacterial development. These findings will allow us to further delve into the study of other bacteria and have a basis for other orthologs containing the HD‐GYP domain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.

Heat Resistance and Salt Hypersensitivity in Lactococcus lactis Due to Spontaneous Mutation of llmg_1816 ( gdpP ) Induced by High-Temperature Growth

During construction of several gene deletion mutants in Lactococcus lactis MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent llmg_1816 mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision. L. lactis Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial L. lactis strain also resulted in spontaneous mutation of llmg_1816 and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of L. lactis and that inactivation of llmg_1816 leads to temperature resistance and salt hypersensitivity.

The exopolyphosphatase TbrPPX1 of Trypanosoma brucei

BackgroundExopolyphosphatases and pyrophosphatases play important but still incompletely understood roles in energy metabolism, and also in other aspects of cell biology such as osmoregulation or signal transduction. Earlier work has suggested that a human exopolyphosphatase, Prune, might exhibit cyclic nucleotide phosphodiesterase activity.ResultsThe kinetoplastida, a large order of unicellular eukaryotes that contains many important pathogens such as Trypanosoma brucei (human sleeping sickness), Trypanosoma cruzi (Chagas disease) or Leishmania ssp (several clinically dinstinct leishmaniases) all contain several exo- and pyrophosphatases. The current study provides a systematic classification of these enzymes, which now allows to situate the information that is already available on some of these enzymes. It then analyses the exopolyphosphatase TbrPPX1 of T. brucei in detail, using RNA interference and genetic knockouts in an attempt to define its function, and immunofluorescence microscopy to study its subcellular localization.TbrPPX1 is an exopolyphosphatase that does hydrolyze pentasodium triphosphate, but not organic triphosphates such as ATP, pyrophosphate or long-chain polyphosphates. Finally, the study investigates the potential cyclic nucleotide phosphodiesterase activity of TbrPPX1.ConclusionsAll kinetoplastid genomes that are currently available contain genes for an exopolyphosphatase and two classes of pyrophosphatases, one associated with the acidocalcisomes and one cytoplasmic. TbrPPX1 represents the T. brucei exopolyphosphatase. It is located throughout the cytoplasm, and its genetic ablation does not produce a dramatic phenotype. Importantly, TbrPPX1 does not exhibit any cyclic nucleotide specific phosphodiesterase activity, which definitively eliminates it as an additional player in cAMP signalling of the kinetoplastida.

Cyclic AMP specific phosphodiesterase activity and colon cancer cell motility.

To investigate mechanisms for regulation of intracellular cAMP involved in cancer cell invasion, phosphodiesterase (PDE) activity in a colon cancer cell line, DLD-1, was studied. Activities of PDE 2, 4, and 5 were detected in DLD-1 cells by pharmacological approach. Specific and cell permeable inhibitors for those PDEs were used to determine which PDE is responsible for cAMP turnover involved in cancer cell motility. Treatment of DLD-1 cells with rolipram and Ro-20-1724 inhibitors for PDE 4, elevated intracellular cAMP contents three to five times of control. EHNA, an inhibitor for PDE 2, and zaprinast. an inhibitor for PDE 5, did not affect cAMP levels. To assess cellular motility, we utilized chemotaxis assay. EHNA and zaprinast did not suppress serum-induced chemotaxis. In contrast, rolipram and Ro-20-1724, suppressed chemotaxis in a dose dependent fashion. These suggest that PDE 4 plays a critical role in regulating intracellular cAMP levels of colon cancer cells and is involved in cancer invasion. PDE 4 can be a novel target of anti-invasion drug.

Sodium chlorate induces undersulfation of cellular proteoglycans and increases in FSH-stimulated estradiol production in immature rat Sertoli cells.

The functional influence of cell proteoglycan (PG) undersulfation on estradiol synthesis by immature rat Sertoli cell cultures was investigated by using sodium chlorate, an inhibitor of the active sulfate donor for sulfotransferases. The addition of sodium chlorate to 20-day-old rat Sertoli cell cultures abolished [35S]-sulfate incorporation into neosynthesized PG and consequently reduced the residence time of undersulfated PG in cell membrane. Simultaneously, follicle-stimulating hormone (FSH)-stimulated estradiol synthesis was increased by 45%. The effects of sodium chlorate upon Sertoli cell PG synthesis and steroidogenesis were not reproduced with the addition of sodium chloride. Addition of phosphodiesterase inhibitors (MIX or Ro20-1724) decreased the magnitude of the chlorate effect on FSH-stimulated steroidogenesis, suggesting that part of chlorate's effect on steroidogenesis resulted from a decrease in adenosine cyclic 3',5'-phosphate (cAMP)-specific phosphodiesterase activity. Additionally, chlorate 1) increased Sertoli cell steroidogenesis at a step located beyond cAMP (restricted to Sertoli cell cultures exhibiting moderate steroidogenic response to (Bu)2cAMP) and 2) abolished the inhibition of steroidogenesis induced by transforming growth factor-beta. These results support our previous data, which showed that alteration in PG synthesis and the consequent decrease in cell membrane PG content induce an increase in FSH-stimulated estradiol synthesis in Sertoli cell cultures. The identification of cAMP-specific phosphodiesterase activity as a signal transduction step modified by PG undersulfation suggests the possible involvement of cell PG in the regulation of phosphodiesterase activity and, therefore, of FSH responsiveness during testicular development.

Changes of Nuclear PI-PLC γ 1 During Rat Liver Regeneration

We have previously demonstrated that rat liver nuclei contain PI-PLC β 1 and γ 1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669–678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP 2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (β 1, γ 1, δ 1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC β 1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear γ 1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC δ 1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC γ 1 that paralleled the in situ observation whereas the β 1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP 2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP 2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the γ 1 isoform of PI-PLC.

The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase.

To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 mumol/L [3H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but not to the rod in bovine retina. ROS-1 immunoadsorbed approximately 80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of approximately 87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.

Peripheral blood mononuclear leukocyte cyclic adenosine monophosphate specific phosphodiesterase activity in childhood atopic dermatitis

Significantly elevated activity of peripheral blood mononuclear cell cAMP specific phosphodiesterase (PDE) activity has been previously reported in adults with atopic dermatitis and in the cord blood of children born to atopic parents. We have been unable to confirm such a significant elevation in children with atopic dermatitis, and we have not found any correlation with dermatitis severity, age or total serum IgE.

Microsomal marker enzymes of Manduca sexta (L) midgut

AbstractThe subcellular distribution of four enzymes (glucose‐6‐phosphatase, phosphodiesterase I, NADPH‐cytochrome c reductase, and p‐nitroanisole O‐demethylase) in the midgut of “wandering” fifth‐instar larvae of the tobacco hornworm, Manduca sexta (L), was determined and the composition of mitochondrial and microsomal pellets was examined by electron microscopy. Most of the glucose‐6‐phosphatase activity and one‐third of the phosphodiesterase I activity were found in the high‐speed supernatant. NADPH‐cytochrome c reductase activity was marginal and O‐demethylase activity was undetectable in the supernatant. The highest specific activities for phosphodiesterase I, NADPH‐cytochrome c reductase, and p‐nitroanisole O‐demethylase were measured in microsomes, but the relative specific activity of phosphodiesterase I was only half that obtained with the latter two enzymes. In all subcellular preparations the relative specific activities of NADPH‐cytochrome c reductase and p‐nitroanisole O‐demethylase were closely correlated. It is concluded that glucose‐6‐phosphatase and phosphodiesterase I are not microsomal marker enzymes in the midgut, but the activities of NADPH‐cytochrome c reductase and p‐nitroanisole O‐demethylase are quantitative measures of microsomal content.

Studies on the regulation of phosphodiesterase in human adipose tissue in vitro.

The influence of several agents known to affect the rate of lipolysis or the phosphodiesterase (PDE) activity in rat adipose tissue was studied in human adipose tissue in vitro. Only insulin, catecholamines, prostaglandin E1, and cAMP increased PDE activity in man (by 20-40% over the control values). In dose-response studies, it was found that the ED50 values for insulin and isopropyl noradrenaline were of the same magnitude as the previously observed ED50 values for lipolysis in human adipose tissue. The time course for insulin and isopropyl noradrenaline showed maximum stimulation after 10 min, followed by a decline, with the curve approaching zero at 60 min. In subcellular fractions of adipose tissue, the highest specific PDE activity was found in the particulate fraction, in which the effects of both insulin and isopropyl noradrenaline were most marked. It is concluded that the stimulatory effect of catecholamines on PDE may be one reason for the failure of these agents to produce a sustained increase in the cAMP level and that the effect of insulin on PDE may be one mechanism by which insulin reduces the rate of lipolysis.

Intracellular Localization of Phosphodiesterase Induced by Cyclic Adenosine 3',5'-Monophosphate in Dictyostelium discoideum

The intracellular distribution of phosphodiesterase [EC 3.1.4.17] induced by cyclic adenosine 3',5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem, J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19 g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17 g/ml) with good reproducibility. Some parts (1.13, 1.17 g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10 g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20,000 X g for 30 min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone. Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.

Isolation of plasma membranes from rabbit myometrium

1. 1. Plasma membranes from the main body of rabbit uterus were prepared by dispersion of smooth muscle cells with collagenase, and disruption with a Parr bomb. 2. 2. [ 125I] Labeled wheat germ agglutinin (WGA) (4–6 μCi//μg) was used to identify plasma membranes. 3. 3. The specific activity (counts/min/mg protein) of [ 125WGA was 9 times higher in the final membrane preparation as compared to the cell homogenate. A similar purification was observed for adenylate cyclase. 4. 4. There was a 5- to 6-fold increase in the specific activity of phosphodiesterase I. 5'-nucleotidase, Mg 2+-dependent ATPase and CaATPase.

3′:5′-Cyclic-AMP Phosphodiesterase Activities in White and Brown Adipose Tissues of Cold-Acclimated Rats

3':5'-Cyclic-AMP phosphodiesterase (PDE) (EC 3.1.4.17) activity was measured in interscapular brown adipose tissue (BAT) and in white epididymal adipose tissue of rats acclimated to constant or fluctuating cold. Experiments were carried out on isolated adipocytes or tissue homogenates. In brown or white adipose tissue or isolated adipocyte homogenates, two different apparent Km values were found according to the substrate (cAMP) concentration. The low Km was at about 10(-6) M and the high one at about 10(-4) M. The apparent V of the high Km enzyme was about 10-fold higher than the V of the low Km enzyme. Cold acclimation to constant or fluctuating cold did not modify appreciably the Km or V values. For low substrate concentrations (10(-6)-10(-8) M), the specific activity of PDE expressed per milligram of protein was decreased in BAT adipocytes of the two groups of cold-acclimated rats, compared to controls. Inversely, it was increased in total tissue homogenates. These variations were smaller in fluctuating cold than in constant cold-acclimate rats. They could, in part, induce the increases in lipolysis and in blood flow observed in the BAT of cold-acclimated rats.

Studies on cyclic AMP phosphodiesterase in sea urchin eggs

1. 1.|Sea urchin eggs have multiple forms of cyclic AMP-phosphodiesterase (EC 3.1.4.17), 70% of which is localized in the post-microsomal supernatant. However, in fertilized eggs at about first cleavage, there is a significant decrease in the amount (55%) of the enzyme detected in the post-microsomal supernatant. This difference in the amount of phosphodiesterase present in the post-microsomal supernatant of cleaving eggs could be accounted for by a 2-fold increase in enzyme activity in heavy membrane fractions of cleaving eggs, as compared to that of unfertilized eggs. 2. 2.|Kinetic studies show two apparent K m values for low and high cyclic AMP concentrations, i.e. 2.5·10 −6 and 1.4·10 −3 M , with V max values of 3.7·10 −8 and 3.2·10 −6 M , respectively. 3. 3.|Mg 2+ is essential for the activity of the enzyme and the optimum pH is 7.60. Dibutyryl cyclic AMP, N 6- monobutyryl cyclic AMP and ADP were found to be inhibitory. Methylxanthines also inhibited the sea urchin enzyme. ATP was found to be the most potent physiological inhibitor of the sea urchin phosphodiesterase. 4. 4.|A 4–5 fold increase in specific activity of phosphodiesterase was obtained in a 30–60% (NH 4) 2SO 4 fraction of the whole homogenate. General properties of the partially purified enzyme were similar to those observed for the whole homogenate enzyme. 5. 5.|Only a very weak cyclic GMP-phosphodiesterase activity was found in these egg-homogenates, which could be due to a non-specific activity of cyclic AMP-phosphodiesterase.