PDEase Activity Research Articles

Phosphomonoesterase and phosphodiesterase activities and their regulation during dinoflagellate blooms under different external phosphate conditions

Phosphatases play a crucial role in recycling organic phosphorus and determining primary production and phytoplankton communities in seawater, especially in phosphate-depleted coastal waters. The present study analyzed spatiotemporal variation in phosphomonoesterase (PMEase) activity (PMEA) and phosphodiesterase (PDEase) activity (PDEA) during 2 dinoflagellate blooms occurring in 2 coastal areas of Fujian Province, East China Sea, which differed in external phosphate conditions. Together with environmental variables and with a specific focus on the availability of different phosphorus forms in the seawater, the regulation of both phosphatases was studied. The results showed that dissolved organic phosphorus (DOP) was the major phosphorus source during blooms, especially in phosphate-depleted environments. Labile DOP accounted for more than 50% of DOP, of which phosphomonoester (PME) and phosphodiester (PDE) accounted for 75-94% and 6-25%, respectively. PMEA and PDEA were highly correlated, both increasing during blooms with a fixed PMEA:PDEA ratio of 2.5 under external phosphate conditions at both sites. Both PMEA and PDEA were negatively correlated with dissolved inorganic phosphorus (DIP) when phosphate was depleted, but positively correlated with DOP regardless of external phosphate conditions. During dinoflagellate blooms, temperature and phytoplankton biomass were the dominant variables determining both phosphatase activities under phosphate-depleted conditions, whereas the availability of DOP was the dominant variable determining both phosphatase activities under phosphate-replete conditions. This study suggests the importance of phosphatases in recycling DOP, and indicates similar regulation of PMEA and PDEA during dinoflagellate blooms.

Characterization of extracellular phosphatase activities in periphytic biofilm from paddy field

Periphytic biofilms exist widely in paddy fields, but their influences on the hydrolysis of organic phosphorus (P) have rarely been investigated. In this study, a periphytic biofilm was incubated in a paddy soil solution, and hydrolysis kinetic parameters (half-saturation constant (Km) and maximum catalytic reaction rate (Vmax)), optimal environmental conditions, substrate specificity, and response to different P regimes of the phosphatase activities in the periphytic biofilm were determined, in order to characterize extracellular phosphatase activities in periphytic biofilms from paddy fields. The results indicated that the periphytic biofilm could produce an acid phosphomonoesterase (PMEase), an alkaline PMEases, and a phosphodiesterase (PDEase). These three phosphatases displayed high substrate affinity, with Km values ranging from 141.03 to 212.96 μmol L–1. The Vmax/Km ratios for the phosphatases followed the order of alkaline PMEase > acid PMEase > PDEase, which suggested that the PMEases, especially the alkaline PMEase, had higher catalytic efficiency. The optimal pH was 6.0 for the acid PMEase activity and 8.0 for the PDEase activity, and the alkaline PMEase activity increased with a pH increase from 7.0 to 12.0. The optimal temperature was 50 °C for the PMEases and 60 °C for the PDEase. The phosphatases showed high catalytic efficiency for condensed P over a wide pH range and for orthophosphate monoesters at pH 11.0, except for inositol hexakisphosphate at pH 6.0. The inorganic P supply was the main factor in the regulation of phosphatase activities. These findings demonstrated that the periphytic biofilm tested had high hydrolysis capacity for organic and condensed P, especially under P-limited conditions.

Phosphatase activities of a microepiphytic community during a bloom of Ostreopsis cf. ovata in the northern Adriatic Sea

It is becoming more apparent that increased organic nutrient loads deriving from anthropogenic activities and natural processes frequently cause the eutrophication of coastal waters. Concurrently, an increasing number of phototrophs have been shown to make use of organic nutrients, mainly through indirect studies of surface enzyme activities or through direct studies of growth in media containing organic-only nutrients. The potential utilization of dissolved organic phosphorus (DOP) by microepiphytic-mats associated with frequently problematic, toxic dinoflagellate Ostreopsis cf. ovata, was investigated throughout a full cycle of a bloom that occurs annually (over the last few decades) during summer along several stretches of the Mediterranean coast. Measurements of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities of the epiphytic mats (including cells and exopolymeric substances) and a range of chemico-physical parameters were made from late summer to early autumn at a eutrophic site.Analyses of ambient nutrient fractions revealed very high aquatic N:P values (TN:TP = 178 ± 50), very low filterable reactive P (FRP) concentrations (13 out 19 under the limits of detection) and DOP concentrations that were on average 85% of the total dissolved P. We recorded a rapid increase in PMEase and PDEase activities in the epiphytic microalgal community that coincided with the onset of a proliferation of the Ostreopsis population. Chromogenic staining of samples showed that activity was closely associated with the Ostreopsis cells, located both extracellularly (cell surface and within the EPS) and intracellularly (ventral cytoplasm). The increase in both phosphatases indicates that Ostreopsis can utilise a wide range of DOP types. The intense activity in the EPS was suggested to aid in the efficient entrapment and processing of high concentration nutrient pulses, for extracellular processing of larger organic materials and to prevent loss of substrates and products to the surrounding water. Based on the present findings, Ostreopsis seems to have adaptations that allow it to thrive in P-limited environments where organic P is the main source of P.

Phosphatase activities in sediments of subtropical lakes with different trophic states

We characterized the vertical distribution of extracellular phosphatase enzymes; phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities in sediments of three subtropical lakes were characterized by different trophic states. We then explored relationships between phosphatase activities, phosphorus (P) compounds, and microbial biomass and activity. Sediment P compounds had been characterized previously by two different methods: sequential fractionation and solution 31P NMR spectroscopy. PMEase and PDEase activities declined with depth and were correlated strongly with microbial biomass and anaerobic respiration, indicating that bacterial phosphatase dominated in these sediments and is an important step in the anaerobic breakdown of organic matter. The oligo-mesotrophic lake had higher PMEase activity and the hypereutrophic lake had higher PDEase activity, while the eutrophic lake had the lowest activities of both enzymes. Principal component analyses showed that enzyme activities were related closely to concentrations of the P forms that they degrade: PMEase activity was correlated with phosphomonoesters, while PDEase activity was correlated with phosphodiesters (including nucleic acids and phospholipids). Enzyme activities were not related to the trophic state but with the concentration P forms found in the sediment. Overall, these results provide insight into the phosphorus cycle in subtropical lake sediments by demonstrating a link between phosphatase activity, P composition, and microbial activity.

CAMP Signaling: An Important Event Facilitating Survival and Infectivity of Leishmania.

IntroductionLeishmania donovani encounters striking shift in temperature and pH and these act as the key environmental trigger for differentiation. Moreover, the parasite also faces a huge oxidative stress inside macrophages. We showed that differentiation condition‐exposed parasites become resistant to oxidative damage due to increased intracellular cAMP‐mediated responses.ObjectiveFor comprehensive understanding of cAMP signaling, we studied all the enzymes associated with cAMP metabolism, viz. the synthesizing enzyme adenylate cyclase, the degrading enzyme, phosphodiesterase (PDE), the regulatory enzyme pyrophosphatase and the functional enzyme, cAMP‐dependent protein kinase (PKA).ResultsA stage‐specific isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation‐coupled induction of cAMP. The membrane bound acidocalcisomal pyrophosphatase, LdV‐H+PPase, was the major isoform regulating cAMP level in association with LdRACA by modulating the total pyrophosphate pool. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defence. Another cytosolic phosphodiesterase, LdPDED though not differentially expressed, was found to down‐regulate cAMP‐mediated responses by inhibiting catalytic activity of PKA and by increasing its own PDEase activity through phosphorylation. Also, a functional cAMP‐binding effector molecule (a regulatory subunit of PKA, LdPKAR) seemed to modulate metacyclogenesis through induction of autophagy.ConclusionThis study reveals the significance of cAMP signaling in parasite survival and infectivity.

Postictal serum nucleotidases activities in patients with epilepsy

Adenosine, a potent anticonvulsant, can be produced in the body by the hydrolysis of adenine nucleotides through the action of ecto- or soluble nucleotidases. Changes in nucleotide hydrolysis occur after pentylenetetrazol-induced epileptic events. We evaluated serum ATP, ADP and AMP hydrolysis rates and soluble nucleotide phosphodiesterase (PDEase) activity at 5, 10, 15, 30 and 60 min, and 12h following an epileptic event. Fifteen patients (seven female, eight male; mean age 15.5 years) were included in the study. The type of seizure was generalized in four patients and was localization related in the remaining 11. There were no differences in adenine nucleotide hydrolysis rates between patients and healthy subjects in the interictal stage. In comparison with controls, ATP, ADP and AMP hydrolysis rates were significantly increased at 5 min (53+/-1.4%, 79.2+/-2.8% and 37.0+/-2.6%, respectively) and up to 30 min following the epileptic event. In contrast to ADP and AMP, ATP hydrolysis remained significantly increased at 60 min (71.4+/-1.6%), returning to the basal level after 12h. Serum PDEase activity was also significantly higher in the patients than in healthy subjects, peaking at 15 min (61+/-2.9%) and remaining significantly increased up to 60 min (4.6+/-1.2%) following the epileptic episode. Globally, the variations in the postictal serum ADP hydrolysis rate almost overlapped those of AMP hydrolysis, whereas changes in the ATP hydrolysis rate overlapped those of PDEase activity. The clinical significance of this elevation in postictal soluble serum nucleotidase activity remains to be clarified. However, it is possible to hypothesize that the higher nucleotidase activity might play a role in the modulation of epileptic events.

Phosphatase activities of the aquatic moss Warnstorfia fluitans (Hedw.) Loeske from an acidic stream in North-East England

A study was made of the aquatic environment, tissue nutrient composition and surface phosphatase activities of the aquatic moss Warnstorfia fluitans in Brandon Pithouse Stream, a small acidic stream in N-E England. The water, which originates from an underground spring, had been pH 2.6 for at least 30 years, but about 3.9 during the present study. The moss was by far the most abundant phototroph during all this period. Seasonal changes in aqueous nitrogen and phosphorus fractions were measured over a 2-year period near the source. Most of the filtrable N and P were at times organic, but the very high N:P ratio (even if organic N is excluded) suggests that only organic phosphate is likely to be important for the moss. There was a high peak in organic phosphate in late spring in both study years. Surface phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities were highly correlated in the field and in axenic culture, though there were some differences in response to environmental factors. Axenic material showed higher PMEase and PDEase activities when grown with organic P than with inorganic P. Although the data suggest that internal P content is an important factor influencing phosphatase activities, PDEase activity was especially marked when the moss was grown with the diester, DNA, as P source, indicating that at least one of its surface phosphatases can also respond directly to the environment.

REGULATION OF PHOSPHATASE ACTIVITY IN CHROOCOCCID- IOPSIS ISOLATES FROM TWO DIVERSE HABITATS: EFFECT OF LIGHT, PH AND TEMPERATURE

Phosphomonoeaterase (PMEase) and phosphodiesterase (PDEase) activity was studied in the cyanobacterial cultures of Chroococcidiopsis isolated from two diverse cryptoendolithic habitats of Antarctic and Arizona. Because this organism is found within the rocks it appears that phosphorus metab- olism by alkaline phosphatase activity is a key factor to sustain growth of these organisms in that state, there being no other source of external P except in bound form in the rocks. The main findings in this paper show that specific pH and temperature regulate the PMEase and PDEase activities studied with different substrates in isolated of Chroococcidiopsis 1 and 2 although values were vastly different. The pH and tem- perature optima for phosphatase activity (PMEase and PDEase) of Chroococcidiopsis 1 and 2 were 9.5, 20 °C and 8.5, 40 °C respectively. It needs mentioning here that although the pH optimum for the enzyme activities in the Antarctic rock samples was the same i.e. 9.5 there was a striking difference in the tem- perature optimum in which maximum activity of both enzymes were recorded at 5 °C. A very crucial role of light and dark conditions were important for the enzyme activity and differed to a significant extent when compared with naturally occurring organisms in the Antarctic rocks. Low light fluxes of about 8 µmol photon m -2 s -1 showed higher PMEase and PDEase activity then total dark conditions in the Chroococcidiopsis -1 culture. However under natural conditions when this organism is found within the rocks 8-µmol photon m -2 s -1 was found to be inhibitory and dark conditions gave higher PMEase and PDEase activity. Arizona rock samples containing Chroococcidiopsis -2 however did not show dark stim- ulation. Increase in light intensity from 8 µmol photon m -2 s -1 to 60 µmol photon m -2 s -1 maintained in the cul- ture room increased the PMEase and PDEase activity of both cultures. The unique light and temperature responses for PMEase and PDEase activities in Chroococcidiopsis-1 found within the Antarctic rocks are unique. It points to some change in the cells probably by producing some cryoprotectant which protects the enzymes from becoming non functional at low temperatures. It also indicates that exposure to light fluxes as low as 8 µmol photon m -2 s

Characterization of the phosphatase activities of mosses in relation to their environment

AbstractPhosphatase activities and environmental features were characterized for 12 terrestrial and aquatic mosses in upland northern England, along with four species sampled from subarctic Sweden. Phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities of shoot tips were measured using para‐nitrophenyl phosphate (p‐NPP) and bis‐pNPP. All species showed PMEase activity, but not all showed PDEase activity. The mean pH optimum was 5·0 for PMEase and 5·7 for PDEase. The kinetic parameters Km and Vmax were calculated from three linear transformations of the Michaelis–Menten equation. The mean Km values of the mosses ranged between 77 and 468 µm for PMEase and 26 and 414 µm for PDEase. The corresponding Vmax values were 0·6–205 µmol pNP g−1 DW h−1 for PMEase and 1·4–110 µmol pNP g−1 DW h−1 for PDEase. Mosses from Sweden displayed greater Km and smaller Vmax values than those from England. The aquatics Fontinalis antipyretica and Rhynchostegium riparioides displayed two‐phase kinetics for PMEase and PDEase, with Km and Vmax being dependent on substrate concentration. Staining suggested that PMEase activity was located in the cell wall of most mosses. Phosphatase assays provide a rapid method for screening environmental nutrient status and a standard procedure is recommended.

Characterization of Fetal Serum 5′-Nucleotide Phosphodiesterase: A Novel Function as a Platelet Aggregation Inhibitor in Fetal Circulation

The study was performed to indicate the ADPase activity of 5′-nucleotide phosphodiesterase (PDE-ase) from human umbilical cord blood serum and demonstrates the effect of this enzyme on ADP-induced platelet aggregation. The PDEase was purified by using p-nitrophenyl-5′-TMP as a substrate. The PDEase had a molecular weight of 128,000 daltons, and activity of 103 nmol/min/mg protein. The PDEase activity was inhibited by 5′-AMP, ADP, ATP. But 2′-AMP, 3′-AMP, 3′:5′ cAMP, and adenosine had no inhibiting effects. Kinetic analysis indicated that ADP was a competitive inhibitor with a K i value of 4.05×10 −5 M. The enzyme was markedly inhibited by 1 mM EDTA. The ADPase activity of the PDEase was 7.79 nmol/min/mg protein. The hydrolized products of ADP by the PDEase were AMP and phosphoric acid. The platelet aggregation by ADP was inhibited by the addition of the PDEase in the platelet-rich plasma.

Alkaline phosphatase activities of anAnabaena sp. from deep-water rice

Anabaena sp. grew with mono- and di-ester phosphate compounds as sources of phosphate, indicating the presence of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities. Cell-bound PMEase and PDEase activities were detected during growth in 0.5 and 10 mg PO4l(-1) only when the cellular phosphate concentration fell to 0.46% of cell protein and the activities increased as cellular phosphate content decreased. The Km values for these enzymes were 0.3MM forp-nitrophenyl phosphate and 0.2MM for bis-p-nitrophenyl phosphate, respectively. Only PMEase activity was found extracellularly. The pH optima for PMEase and PDEase were 10.2 and 10.4, respectively, and the temperature optima at pH 10.2 were 37°C and 40°C, respectively. Ca(2+) increased the enzyme activities while Zn(2+) caused marked inhibition. The inorganic phosphate repressed the cellular PMEase activity after a lag of 4 h.

Improved cytochemical method for cyclic 3',5'-nucleotide phosphodiesterase in rat rod outer segments.

It is of increasing interest to observe the precise localization of cyclic 3′, 5′-nucleotide phosphodiesterase (PDEase) activity in rod outer segments at the ultrastructural level. The cytochemical method of PDEase localization at the ultrastructural level was developed by Florendo et al. However, there were two major difficulties in the use of snake venom as an exogenous 5′-Nucleotidase (5′-Nase): 1) the marked ultrastructural damage due to snake venom, and 2) the inadequate penetration of exogenous 5′-Nase into tissues. Therefore, two improvements for the demonstration of PDEase activity were carried out by the case of: 1) a purified 5′-Nase as exogenous 5′-Nase for a better preservation of cell morphology, and 2) 40μm sections made with the use of a freezing microtome for a better penetration of 5′-Nase into the tissues.PDEase activity using purified 5′-Nase was observed along the disc in the rod outer segment, and no retinal detachment was observed between the outer segments and pigment epithelium, the ultrastructure being excellently preserved, as compared with the use of snake venom. By applying this improved method to freeze-substitution technique, the catalytic site of PDEase was localized on the cytoplasmic surface of the disc membranes. This new technique would be useful in detecting the precise localization of PDEase activity.

Characterization of the memory gene dunce of Drosophila melanogaster

The dunce ( dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4·2 to 9·6 kb (1 kb = 10 3 bases or base-pairs). To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs. Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes. Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites; two of these were mapped by parallel S 1-nuclease and primer extension experiments. In addition, some of the heterogeneity is generated by using varying lengths of a 3′-untranslated trailer sequence. Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3′ ends. The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5′ exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3′ splice sites separated by six base-pairs. Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation. The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase.

Phosphatase activity of the blue-green alga (cyanobacterium) Nostoc commune UTEX 584

Abstract Nostoc commune UTEX 584 was observed to grow on a range of organic phosphate sources, indicating the possession of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities, but it was unable to utilize phytic acid. The use of ρ-nitrophenyl phosphate (ρNPP) as substrate showed the presence of cell-bound and extracellular PMEase. Cell-bound PMEase and PDEase were detected during growth in batch cultures, when the mean cellular phosphate content fell to 0.76% algal dry weight. Use of bis-ρ-nitrophenyl phosphate (bis-ρNPP) showed that cell-bound PDEase activity (measured as ρ-nitrophenol released) was about half that of PMEase, but that no extracellular PDEase activity was present. PMEase and PDEase activities both decreased when the cellular phosphate content fell below 0.3%. The optimum temperatures for PMEase and PDEase activity at pH 7.6 were 32°C and 42°C, respectively; the optima were lower when the assay was performed at pH 10.3. The pH optima for PMEase and PDEase differed sligh...

Cloning and Expression of cDNA for a Human Low-Km, Rolipram-Sensitive Cyclic AMP Phosphodiesterase

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

The Effects of Mercuric Chloride on Calmodulin-Mediated CA+2 Transport in Rat Brain

We have shown previously that mercuric chloride (HgCl2) inhibits in vitro vasopressin release from the isolated rat neurohypophysis with maximum inhibition occurring with 0.5 mM HgCl2. Associated with the inhibition of hormone release is an increase in 45Ca+2 uptake, an increase in cytosolic 45Ca+2, and a reduction of 45Ca+2 accumulation by mitochondria in the intact gland. In the present series of studies, the effect of HgCl2 on calmodulin (CM) function in neural tissue preparations is reported. Mercuric chloride (0.5 mM) reduced 45Ca+2 binding to CM purified from bovine neurohypophyses by 20% and inhibited endogenous CM-stimulated Ca,Mg-ATPase activity from rat brain mitochondria in a dose-dependent fashion. Ca,Mg-ATPase activity was inhibited by 50 and 80% with 0.5 and 5.0 mM HgCl2, respectively. CM-stimulation of Ca,Mg-ATPase activity was inhibited by calmidazolium (CMZ) with maximal inhibition seen with 0.1 mM CMZ. Reversibility of the HgCl2 interaction with CM was demonstrated using CM-stimulated phosphodiesterase (PDEase) activity from rat brain. HgCl2 inhibited both basal and CM-stimulated PDEase activity in a dose-dependent manner with maximum inhibition occurring with 1.0 mM HgCl2. Preexposure of CM to an inhibitory concentration (1.0 mM) of HgCl2 resulted in no loss of stimulatory PDEase enzyme activity. From these results, we conclude that HgCl2 reversibly interferes with 45Ca+2 binding to CM and also inhibits CM-regulated Ca+2 pumping enzyme systems in the neurohypophysis. The inhibition of vasopressin release from the intact gland in the presence of HgCl2 thus, may be associated with a disruption of calcium in the neurohypophysis.

Human salivary gustin is a potent activator of calmodulin-dependent brain phosphodiesterase.

Human salivary gustin stimulated activity of brain calmodulin-dependent cyclic nucleotide phosphodiesterase (cAMP PDEase; 3',5'-cyclic-nucleotide phosphodiesterase, EC 3.1.4.17) in a dose-dependent manner in the absence of calmodulin. At physiological levels found in human saliva, gustin activated cAMP PDEase 5- to 6-fold. Activation of PDEase occurred with as little as 500 ng of gustin. Comparative sensitivity of activation of PDEase by gustin was intermediate between calmodulin and lysophosphatidylcholine with maximal activation and half-maximal activation (indicated in parentheses) at 3 X 10(-8) M (4.3 X 10(-9) M), 3.4 X 10(-6) M (3.4 X 10(-7) M), and 2.5 X 10(-3) M (4.0 X 10(-5) M) for calmodulin, gustin, and lysophosphatidylcholine, respectively. No other major salivary protein activated PDEase. Anticalmodulin antibody completely inhibited calmodulin-activated cAMP PDEase activity, but the antibody had no effect on gustin-activated cAMP PDEase activity. A sensitive calmodulin RIA indicated that no calmodulin was detected in any gustin preparation that activated cAMP PDEase. Both gustin and calmodulin rendered cAMP PDEase thermally labile to a similar extent and increased Vmax without affecting the apparent Km for the substrate cAMP. Activation by gustin and calmodulin was unaffected by lubrol-PX, trypsin inhibitor, pepstatin A, or leupeptin. In the presence of 1 mM EGTA, gustin activated cAMP PDE 5- to 6-fold, but the activating ability was completely lost after gustin was heated at 100 degrees C for 5 min. In contrast, calmodulin lost all activating ability in the presence of 1 mM EGTA, whereas heating calmodulin at 100 degrees C for 5 min did not affect its activation of cAMP PDEase. Lysophosphatidylcholine-activation of cAMP PDEase, like gustin activation, was unaffected by EGTA, but lysophosphatidylcholine-activation of cAMP PDEase, like calmodulin activation, was unaffected by heating at 100 degrees C for 5 min.

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)

Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cGMP binding sites of rod phosphodiesterase.

In illuminated rod outer segment membranes, GTP and guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) have reciprocal effects on cGMP phosphodiesterase (PDEase; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) activity and cGMP binding to noncatalytic sites on that enzyme. Two micromolar p[NH]ppG increased PDEase activity more than 2-fold while inhibiting cGMP binding more than 40%. Reduction of noncatalytic cGMP binding, which followed addition of p[NH]ppG, was not a result of PDEase activation. Both effects of p[NH]ppG were completely dependent on the presence of bleached rhodopsin. A heat-stable factor has been found to inhibit PDEase activity and also to stimulate cGMP binding to noncatalytic cGMP binding sites. Addition of p[NH]ppG reversed the effects of this factor on both PDEase activity and cGMP binding. During purification of this material, the activity peaks for both PDEase inhibition and activation of noncatalytic cGMP binding comigrated on both Blue Sepharose CL-6B column chromatography and sucrose density gradients centrifugation, suggesting that the same factor could be responsible for both inhibition of PDEase activity and enhancement of noncatalytic cGMP binding. Limited tryptic proteolysis of PDEase, which markedly reduced cGMP binding to the noncatalytic sites, and experiments using highly purified cAMP (free of cGMP) as substrate for PDEase showed that the binding of cGMP to noncatalytic sites was not required for the heat-stable inhibitory factor to inhibit PDEase activity. We discuss possible relationships between the regulation of PDEase and the binding of cGMP to noncatalytic sites.